U.S. patent application number 17/226739 was filed with the patent office on 2021-12-02 for compounds.
The applicant listed for this patent is MedImmune Limited. Invention is credited to Simon Charles Cruwys, Steven Godfrey Lane, Philip Mallinder.
Application Number | 20210371513 17/226739 |
Document ID | / |
Family ID | 1000005770074 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210371513 |
Kind Code |
A1 |
Cruwys; Simon Charles ; et
al. |
December 2, 2021 |
Compounds
Abstract
Binding members, e.g. human antibody molecules, which bind
interleukin-6 (IL-6) and neutralise its biological effects. Use of
binding members for IL-6 in medical treatment e.g. for treating
inflammatory diseases and tumours associated with IL-6.
Inventors: |
Cruwys; Simon Charles;
(Ewell, GB) ; Lane; Steven Godfrey; (Cambridge,
GB) ; Mallinder; Philip; (Loughborough, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune Limited |
Cambridge |
|
GB |
|
|
Family ID: |
1000005770074 |
Appl. No.: |
17/226739 |
Filed: |
April 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16361062 |
Mar 21, 2019 |
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17226739 |
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15639949 |
Jun 30, 2017 |
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16361062 |
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14675980 |
Apr 1, 2015 |
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15639949 |
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13480690 |
May 25, 2012 |
9005620 |
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14675980 |
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11948659 |
Nov 30, 2007 |
8198414 |
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13480690 |
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60861704 |
Nov 30, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
A61K 2039/505 20130101; C07K 2317/21 20130101; C07K 16/248
20130101; C07K 2317/34 20130101; C07K 2317/51 20130101; C07K
2317/56 20130101; C07K 2317/73 20130101; C07K 2317/515 20130101;
G01N 33/6857 20130101; C07K 2317/92 20130101; C07K 2317/567
20130101; C07K 2317/622 20130101; G01N 2333/5412 20130101; C07K
2317/76 20130101; C07K 2317/565 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; G01N 33/68 20060101 G01N033/68 |
Claims
1. An isolated binding member for human IL-6, which binds human
IL-6 with a K.sub.D of not more than 30 pM as defined by surface
plasmon resonance.
2-22. (canceled)
23. An isolated binding member for human IL-6, comprising an HCDR3,
an LCDR3 and/or a set of CDRs of any of antibodies 2, 3, 4, 5, 7,
8, 10, 14, 16, 17, 18, 19, 21, 22 or 23.
24-58. (canceled)
59. A method for producing an antibody antigen-binding domain for
IL-6, the method comprising providing, by way of addition,
deletion, substitution or insertion of one or more amino acids in
the amino acid sequence of a parent VH domain comprising HCDR1,
HCDR2 and HCDR3, wherein the parent VH domain HCDR1, HCDR2 and
HCDR3 are a set of HCDRs as shown in Table 7, a VH domain which is
an amino acid sequence variant of the parent VH domain, and
optionally combining the VH domain thus provided with one or more
VL domains to provide one or more VH/VL combinations; and testing
said VH domain which is an amino acid sequence variant of the
parent VH domain or the VH/VL combination or combinations to
identify an antibody antigen binding domain for IL-6.
60-73. (canceled)
Description
[0001] This application is a continuation of U.S. application Ser.
No. 16/361,062, filed Mar. 21, 2019, which is a continuation of
U.S. application Ser. No. 15/639,949, filed Jun. 30, 2017, which is
a continuation of U.S. application Ser. No. 14/675,980, filed Apr.
1, 2015, which is a continuation of U.S. application Ser. No.
13/480,690, filed May 25, 2012, now U.S. Pat. No. 9,005,620, which
is a continuation of Ser. No. 11/948,659, filed Nov. 30, 2007, now
U.S. Pat. No. 8,198,414, which claims priority to U.S. Provisional
Application No. 60/861,704, filed Nov. 30, 2006, the disclosures of
which are hereby incorporated by reference in their entirety.
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy created on Apr. 9, 2021,
is named 48519US_CRF_sequencelisting.txt and is 108,614 bytes in
size.
[0003] This invention relates to binding members, especially
antibody molecules, which inhibit biological effects of IL-6. The
binding members are useful for treatment of disorders associated
with IL-6, including inflammatory diseases and tumours.
[0004] Interleukin 6 (IL-6) is a 26 kDa pleiotropic
pro-inflammatory cytokine produced by a variety of cell types,
including stimulated fibroblasts, monocytes and endothelial cells,
which form the major source of IL-6 in vivo. Cells such as T cells,
B cells, macrophages, keratinocytes, osteoblasts and several others
can produce IL-6 on stimulation. IL-6 is also expressed from tumour
cell lines and tumour cells e.g. cells from lung carcinoma,
prostate cancer, myeloma, hypernephroma and cardiac myxoma [1, 2].
Under non-inflammatory conditions, IL-6 is secreted from adipose
tissue [3].
[0005] The regulation of IL-6 expression depends on the cell type
that is producing it. In multiple myeloma cells IL-6 appears to act
in a positive feedback loop--stimulating the cells to grow as well
as produce more IL-6 [4, 5]. In other cell types IL-6 appears to
inhibit the growth and activation of cells and may act as a
negative regulator for some pro-inflammatory cytokines.
[0006] To initiate cell signalling, IL-6 binds with low affinity to
a transmembrane receptor, IL-6 receptor alpha (also referred to as
IL-6R.alpha., IL-6Ra, IL-6R, gp80 or CD126) to form a complex
"IL-6:IL-6Ra". This complex binds to the gp130 signal receptor;
IL-6R.alpha. and gp130 together form a high affinity IL-6 binding
site, and induce the formation of a hexamer composed of two copies
each of IL-6, IL-6Ra and gp130 [6]. The transmembrane and
cytoplasmic domains of the IL-6Ra are not required for signal
transduction, as IL-6Ra also exists as a soluble secreted form
(sIL-6R or sIL-6Ra). The soluble receptor is produced either by
differential splicing of the IL-6Ra message or by proteolytic
shedding. sIL-6R is capable of forming a ligand-receptor complex
with IL-6, "IL-6:sIL-6Ra". This complex can bind gp130 on cells and
thereby initiate cell signalling in gp130 positive cells, even if
those cells do not express IL-6Ra. Thus, sIL-6R has the potential
to widen the repertoire of cells responsive to IL-6, and is thought
to play an important role in IL-6-mediated inflammation [7].
[0007] A crystal structure of human IL-6 ligand has been elucidated
[6]. The crystal structure of the extracellular domain of human
IL-6Ra [8], and the hexameric structure of IL-6/IL-6R/gp130 complex
[9], have also been resolved. These structures combined with
mutagenesis studies have identified three sites on the surface of
IL-6 which are involved in the functional activity of the IL-6 in
complex with the various receptor components. Site 1 residues are
involved in the interaction between IL-6 and IL-6Ra. Site 2
residues are involved in the interaction between IL-6 and the gp130
cytokine binding domain. The residues in Site 3 of IL-6 are
involved in interacting with the Ig-like domain of the second gp130
in the hexameric complex. A fourth site on IL-6 has also been
identified where IL-6 interacts with the second molecule of IL-6 in
the hexameric IL-6/IL-6R/gp130 complex [10].
[0008] A number of anti-IL-6 ligand monoclonal antibodies have been
isolated. Mapping studies have been performed which show that these
bind to different binding sites, as described above, on the surface
of human IL-6 [11, 12, 13, 14, 15].
[0009] A number of anti-IL-6Ra monoclonal antibodies have also been
generated and their binding sites on the IL-6Ra mapped [16, 14, 15,
17].
[0010] IL-6 belongs to a family of cytokines, which includes
Interleukin-11 (IL-11), ciliary neurotrophic factor (CNTF),
Oncostatin M (OsM), Leukaemia Inhibitory Factor (LIF),
cardiotrophin-like cytokine (CLC), and Cardiotrophin 1 (CT-1). Each
of the members of this family have their own specific receptor
alpha subunits and form complexes with the common receptor subunit
gp130. Targeted disruption of the gp130 gene is embryonically
lethal [18, 19]. All members of the IL-6 family can induce the
expression of acute phase proteins from hepatocytes.
[0011] IL-6 signalling involves tyrosine phosphorylation by JAK
family kinases, and subsequent activation of two major
intracellular signalling cascades, the SHP2/ERK MAPK and STAT1/3
pathways, leading to gene expression via NF-IL-6 and AP-1 [18,
20].
[0012] IL-6 shows a wide spectrum of biological functions
including: haematopoiesis, induction of acute phase responses, T
cell activation, stimulation of antibody secretion, host defence
against infection, myeloma cell and osteoclast activation [21, 22].
For a review of the effects of IL-6 see ref. [23]. IL-6 was
originally identified as a B-cell differentiation factor generated
by T cells [24] but has subsequently been identified as a potent
activator and growth-promoting factor of many cell types. It
induces the final maturation of B cells into antibody producing
cells and is an essential accessory factor for T cell activation
and proliferation. Studies have shown that IL-6 is involved in the
activation of auto-reactive T lymphocytes and the proliferation and
differentiation of cytotoxic T cells. IL-6 has been implicated in
haematopoiesis as a cofactor causing the activation and
differentiation of haemopoietic stem cells. The effect of IL-6 on
the acute phase response is also well documented [25]. IL-6 induces
a variety of acute phase proteins including fibrinogen,
alpha-anti-chymotrypsin, serum amyloid A and C-reactive protein
from human hepatocytes. Acute phase proteins control immune
responses and inflammation and have effects on tissue remodelling.
The serum level of IL-6 correlates well with that of C-reactive
protein in variety of pathologies suggesting a causal role of IL-6
in the acute phase response. IL-6 has also been shown to be
produced by osteoblasts and appears to be involved in osteoclast
activation and bone resorption [26, 27, 28]. Paradoxically it has
been suggested that IL-6 not only has roles as a pro-inflammatory
cytokine but can also, in certain circumstances and cell types,
dampen the effects of other pro-inflammatory cytokines leading to a
reduction in inflammation.
[0013] Because IL-6 has a variety of biological effects, the
elevation of IL-6 has been implicated as a key cytokine in a
variety of disease indications. The levels of circulating IL-6 have
been shown to be elevated in diseases such as rheumatoid arthritis,
Castleman's disease, Juvenile idiopathic arthritis and Crohn's
Disease [29]. Because of this IL-6 has been implicated in driving
the pathology in these inflammatory indications. Furthermore, a
variety of tumour types have been shown to be stimulated by IL-6,
including melanoma, renal cell carcinoma, Kaposi's sarcoma, ovarian
carcinoma, lymphoma, leukaemia, multiple myeloma, and prostate
carcinoma [30]. Moreover increased circulating levels of IL-6 have
been reported in several cancers. In some cancer indications
elevated IL-6 levels has been used as prognostic indicators of the
disease.
[0014] Because of the role of IL-6 in disease a variety of murine
and chimeric anti-human IL-6 monoclonal antibodies have been
developed as potential therapies.
[0015] U.S. Pat. No. 5,856,135 describes a reshaped human antibody
to IL-6, derived from a mouse monoclonal antibody "SK2".
JP-10-66582 reports a chimeric antibody to IL-6, which is indicated
as recognising the helix D region of IL-6 (site 1).
[0016] WO2004/020633 (EP1536012) describes a human scFv antibody
molecule to IL-6 isolated using phage display technology. The scFv
is reported to have an affinity of 13 nM.
[0017] A murine anti-IL-6 antibody, elsilimomab (also known as
B-E8) has been used to treat patients with Multiple myeloma [31,
32] renal cell carcinoma [33] and rheumatoid arthritis [34] and
improvements in certain diagnostic markers were seen in treated
patients with all three diseases. BE-8 has also been used to treat
HIV-positive patients with immunoblastic or polymorphic large cell
lymphoma [35] with relief of systemic symptoms (i.e. fever, sweats,
cachexia) and suppression of spontaneous growth of the lymphoma in
approximately 50% of patients.
[0018] However, the rapid clearance of this antibody and possible
anaphylactic reactions due to the production of human anti-mouse
antibodies (HAMA) to elsilimomab has limited its use in the clinic
[36].
[0019] In general, clinical use of murine monoclonal antibodies is
limited, as such antibodies frequently induce HAMA. HAMA directed
against the Fc part of the mouse immunoglobulin are often produced,
resulting in rapid clearance of anti-IL-6 mAb and possible
anaphylactic reaction [36]. It is also known that the
pharmacokinetics of mouse antibodies in humans is different from
human antibodies having shorter half lives and increased rates of
clearance.
[0020] To reduce the immunogenicity of murine antibodies in humans,
chimeric antibodies with mouse variable regions and human constant
regions have been constructed. A chimeric human-mouse anti-IL-6
antibody cCLB8 (known as CNTO 328) has been used to treat patients
with multiple myeloma [5, 37], with disease stabilisation seen in
the majority of patients.
[0021] However, although chimeric antibodies are less immunogenic
than murine MAbs, human anti-chimeric antibody (HACAs) responses
have been reported [38].
[0022] Mapping studies on cCLB8 have been carried out which show it
is a site I inhibitor of IL-6 activity. Brakenhoff et al [39]
demonstrated that cCLB8 binds to IL-6 amino-terminal deletion
mutants Pro46, Ser49, Glu51, Ile53, Asp54 and also binds to
deletion mutants Asp62 and Met77 (albeit at reduced affinity). The
same authors show that cCLB8 inhibits wild type IL-6 but not
C-terminal deletion 5 in a B9 cell proliferation assay and that
cCLB8 will not bind IL-6 del C-4 which has the last 4 C-terminal
amino acids residues deleted. This data suggest that cCLB8 binds to
an epitope involving the C-terminal residues of IL-6.
[0023] Kalai et al [17] demonstrated that cCLB8 failed to recognise
IL-6 mutants F106E, F102E/F106E or R207E/R210E. However the
antibody does recognise IL-6 mutants R207E and R207W. The binding
of cCLB8 to mutants R207W & R207E is approximately 50% of that
compared to wild type, which suggests that residues F106 and R210
are involved in the cCLB8 binding epitope and residue R207 is
involved in binding but has less effect than residues F106 and
R210. The cCLB8 binds IL-6 site-I mutants R196M, K199N/Q203L and
Q203L with 100% activity compared to wild type. Brakenhoff et al
[13] demonstrated that cCLB8 binds the following IL-6 variants;
Q182H, N183K, W185Q, W185G, W185R, T190P, Q182H/Q184P, W185R/S197N,
Q187E/T190P, 1164L/L186R/M1891, which is not surprising as the
majority of these are distally separated from the IL-6 site 1
residues.
[0024] The positive effect of inhibiting IL-6 signalling in cancer
and inflammatory diseases has been further highlighted by the use
of a humanised anti-IL-6Ra antibody Tocilizumab (also known as
hPM-1, MRA and Actemra). This is a humanised version of the murine
anti-IL6Ra antibody PM-1. Treatment of patients with this antibody
has proven effective in a number of diseases including rheumatoid
arthritis, Juvenile idiopathic arthritis, Crohn's disease,
Myeloproliferative disorder, Castleman's disease and Systemic lupus
erythematosus [40].
[0025] We have succeeded in isolating highly potent, high affinity
binding members for IL-6. Owing to their high affinity and potency,
and their performance in functional studies as described herein,
binding members of the invention are particularly suitable for use
in therapeutic and/or diagnostic treatment of the human or animal
body.
[0026] The binding members are useful for treating disorders
associated with IL-6, as described in detail elsewhere herein.
[0027] A human anti-IL-6 antibody for the treatment of inflammatory
diseases and cancer provides significant advantages over existing
approaches. For example, human antibodies do not induce HAMA or
HACA responses, and have a longer in vivo half life compared with
non-human or chimeric antibodies.
[0028] We have also recognised that binding members for IL-6 offer
significant advantages as compared with binding members for IL-6Ra,
especially in terms of in vivo administration and treatment, as
described elsewhere herein.
[0029] As described in more detail in the Examples, we isolated a
parent antibody molecule, designated CAN022D10, with a set of CDR
sequences as shown in Table 7. Through a process of optimisation we
generated a panel of antibody clones: Antibodies 2, 3, 4, 5, 7, 8,
10, 14, 16, 17, 18, 19, 21, 22 and 23, with CDR sequences derived
from the parent CDR sequences and having substitutions at the
positions indicated in Table 7.
[0030] Thus for example it can be seen from Table 7 that Antibody 2
has a parent HCDR1 sequence in which Kabat residue 35 is replaced
with Thr (SEQ ID NO: 13). Antibodies 14 and 22 contain an
additional residue, i.e. an amino acid insertion, in HCDR3: Ile at
Kabat residue 100D, which is not present in the parent HCDR3
sequence SEQ ID NO: 5. Antibodies 7, 8, 10, 16-19, 21 and 23 do not
contain Kabat residue 95 in LCDR3, whereas the parent LCDR3 (SEQ ID
NO: 10) comprises Pro at Kabat residue 95. The parent HCDR3, and
HCDR3 sequences of all of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16,
17, 18, 19, 21, 22 and 23 have Trp at Kabat residue 95 and Asp at
Kabat residue 101, indicating that H95 Trp and H101 Asp may
contribute to binding and/or potency for IL-6 in binding members of
the invention.
[0031] VH domain, VL domain and CDR sequences of the parent
antibody CAN022D10, and of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16,
17, 18, 19, 21, 22 and 23 as described herein are shown in the
appended sequence listing.
[0032] As described in more detail below, binding members according
to the invention have been shown to neutralise IL-6 with high
potency. Neutralisation means inhibition of a biological activity
of IL-6. Binding members of the invention may neutralise one or
more activities of IL-6. The inhibited biological activity is
typically IL-6 binding to one or more of its binding partners. For
example, the inhibited biological activity may be binding of IL-6
to transmembrane and/or soluble IL-6R.alpha.. This is demonstrated
in the following assays, which are described briefly here and in
more detail below: The TF-1 assay shows that binding members
according to the invention inhibit IL-6 binding to membrane IL-6Ra
as the TF-1 cells do not appear to produce soluble IL-6Ra. As such,
the binding members of the invention therefore inhibit IL-6 binding
to the membrane receptor. In the synovial fibroblast assay, binding
members according to the invention inhibit IL-6 binding to soluble
IL-6Ra since sIL-6Ra needs to be added to this assay for it to
work. The added IL-1beta induces production of endogenous IL-6
which when inhibited by a binding member of this invention prevents
VEGF production.
[0033] In accordance with the invention, binding of human or
non-human primate, e.g. cynomolgus, IL-6 to IL-6R.alpha. may be
inhibited, e.g. a binding member may inhibit binding of mature
human IL-6 to IL-6R.alpha..
[0034] Inhibition in biological activity may be partial or total.
Binding members may inhibit IL-6 biological activity by 100%, or at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the binding member.
[0035] Neutralising potency of a binding member may be determined.
Potency is normally expressed as an IC.sub.50 value, in nM unless
otherwise stated. In functional assays, IC.sub.50 is the
concentration of a binding member that reduces a biological
response by 50% of its maximum. In ligand-binding studies,
IC.sub.50 is the concentration that reduces formation of the
ligand-receptor complex by 50% of the maximal specific binding
level. IC.sub.50 may be calculated by plotting % of maximal
biological response as a function of the log of the binding member
concentration, and using a software program, such as Prism
(GraphPad) or Origin (Origin Labs) to fit a sigmoidal function to
the data to generate IC.sub.50 values. Potency may be determined or
measured using one or more assays known to the skilled person
and/or as described or referred to herein.
[0036] Neutralisation of IL-6 activity by a binding member in an
assay described herein, e.g. the TF-1 proliferation assay or other
cell-based assays described below, indicates that the binding
member binds and neutralises IL-6. Other methods that may be used
for determining binding of a binding member to IL-6 include ELISA,
Western blotting, immunoprecipitation, affinity chromatography and
biochemical assays.
[0037] Binding members described herein were demonstrated to bind
and neutralise biological effects of endogenous human IL-6, as
shown in an assay of inhibition of VEGF release from human synovial
fibroblasts in response to endogenous human IL-6, reported in
Examples 1.7 and 2.7 herein. In this assay, synovial fibroblasts
from rheumatoid arthritis patients produce IL-6 in response to
stimulation with IL-1.beta. and soluble IL-6R.alpha., leading to
IL-6 induced secretion of VEGF. The IL-6 produced by the human
synovial fibroblasts thus represents endogenous human IL-6.
Endogenous IL-6 is the molecular target for medical treatment in
humans, so neutralisation of endogenous IL-6 is an important
indicator of the therapeutic potential of the binding members.
Since the assays were conducted with synovial fibroblasts obtained
from rheumatoid arthritis patients, the results are particularly
relevant to use of the binding members for treating rheumatoid
arthritis. Neutralising potency of optimised antibody molecules
tested in the VEGF release assay surpassed that of the known anti
IL-6 antibody CNTO-328.
[0038] A binding member according to the invention may have an
IC.sub.50 of less than 50 nM, e.g. less than 5 nM, e.g. less than 1
nM in an assay of inhibition of VEGF release from human synovial
fibroblasts stimulated with 0.6 pM human IL-1.beta. and 2.4 nM
soluble human IL-6R.alpha..
[0039] Endogenous IL-6 is known to be a mixture of glycosylated and
unglycosylated forms. Binding of a binding member of the invention
to endogenous IL-6 has been demonstrated in the synovial fibroblast
assay since this assay utilises IL-6 from human synovial
fibroblasts i.e. endogenous IL-6.
[0040] A binding member of the invention may inhibit IL-6 induced
proliferation of TF-1 cells. TF-1 is a human premyeloid cell line
established from a patient with erythroleukaemia (Kitamura et al
1989). The TF-1 cell line requires the presence of a growth factor
for survival and proliferation. The individual growth factors TF-1
cells can respond to include IL-6, GM-CSF and Oncostatin M. A
binding member of the invention may have an IC.sub.50 of less than
100 nM, e.g. less than 20 nM, 10 nM or 1 nM, e.g. less than 100 pM,
70 pM, 50 pM, 40 pM, 30 pM, 20 pM or 10 pM, in an assay for
inhibition of proliferation of TF-1 cells in response to 20 pM
human IL-6. As described herein (see Example 1.5), a parent IgG
"CAN022D10" was shown to have an IC50 in the TF-1 proliferation
assay of about 93 nM, and we subsequently generated optimised
variants of CAN022D10 having substantially increased potency
(IC.sub.50 generally less than 100 pM), as shown in Examples 2.2,
2.5 and 2.6 (Tables 3, 4 and 5, respectively). Notably, IC.sub.50
values for some of the optimised clones were measured to be low as
5 pM or less, for example the germlined IgG Antibody 7, Antibody 17
and Antibody 18, representing extremely high neutralising potency
of these antibodies.
[0041] A binding member of the invention may inhibit IL-6 induced
proliferation of B9 cells. B9 cells are a sub-clone of the murine
B-cell hybridoma cell line, B13.29, selected on the basis of their
specific response to IL-6. B9 cells require IL-6 for survival and
proliferation and respond to very low concentrations of IL-6. As
such, proliferation of these cells in the presence of an IL-6
antibody can be assessed and the affinity of the antibody
determined. Example 2.10 herein shows that Antibody 18 inhibited B9
cell proliferation in response to IL-6, and showed high affinity in
this assay.
[0042] Auto-antibody production in rheumatoid arthritis is mostly
of the IgM class. SKW6.4 is a clonal IgM secreting human
lymphoblastoid B cell line. Upon stimulation with IL-6 these cells
secrete IgM, thus this assay was perceived to be relevant to
rheumatoid arthritis. SKW6.4 cells may be used in an assay to
determine potency of binding members for neutralising IL-6, by
determining inhibition of IgM secretion in response to IL-6. A
binding member of the invention may have an IC50 of less than 10
pM, e.g. less than 5 pM, in an SKW6.4 cell assay of inhibition of
IgM secretion in response to 100 pM human IL-6. Antibody 18 was
shown to neutralise effects of IL-6 in this assay--see Example 2.11
(Table 9).
[0043] The invention provides high affinity binding members for
human IL-6. High affinity for IL-6 from cynomolgus monkey was also
demonstrated. A binding member of the invention may bind human IL-6
and/or cynomolgus IL-6 with a K.sub.D of not more than 1 nM, e.g.
not more than 100 pM, 50 pM, 30 pM or 10 pM. The K.sub.D may be
determined by surface plasmon resonance, e.g. BIAcore.RTM..
BIAcore.RTM. measurements of affinity are described herein in
Example 2.9. Remarkably, the affinity of Antibodies 7 and 18 was
found to be beyond the limit measurable using the BIAcore.RTM.
instrument, indicating a K.sub.D value below 10 pM.
[0044] As described elsewhere herein, surface plasmon resonance
involves passing an analyte in fluid phase over a ligand attached
to a support, and determining binding between analyte and ligand.
Surface plasmon resonance may for example be performed whereby IL-6
is passed in fluid phase over a binding member attached to a
support. Surface plasmon resonance data may be fitted to a
monovalent analyte data model. An affinity constant Kd may be
calculated from the ratio of rate constants kd/ka as determined by
surface plasmon resonance using a monovalent analyte data
model.
[0045] Affinity of a binding member for IL-16 may alternatively be
calculated by Schild analysis, e.g. based on an assay of inhibition
of TF-1 cell proliferation in response to varied concentrations of
human IL-6. A binding member of the invention may have an affinity
of less than 10 pM, e.g. less than 1 pM, as calculated by Schild
analysis. As reported in Example 2.10 herein, the affinity of
Antibody 18 for human IL-6 was calculated as 0.4 pM using Schild
analysis.
[0046] A binding member of the invention may optionally not
cross-react with one or more, or all, of the following: leukaemia
inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), IL-11
or oncostatin M.
[0047] A binding member of the invention may optionally not
cross-react with rat IL-6, mouse IL-6 and/or dog IL-6.
[0048] Cross-reactivity of binding members for binding other
proteins or non-human IL-6 may be tested for example in a time
resolved fluorescence assay for inhibition of human IL-6 binding to
the binding member immobilised on a support, such as the
DELFIA.RTM. epitope competition assay as described in Example 1.6.
For example, any or all of LIF, CNTF, IL-11, oncostatin M, rat IL-6
and mouse IL-6 may show no inhibition, less than 50% inhibition, or
may have an IC.sub.50 greater than 0.5 mM or greater than 1 mM in
the time resolved fluorescence assay for inhibition of labelled
human IL-6 binding to the binding member immobilised on a support.
For example, any or all of LIF, CNTF, IL-11, oncostatin M, rat IL-6
and mouse IL-6 may show no inhibition or may have an IC.sub.50 at
least 10- or 100-fold greater than that of unlabeled human IL-6 in
the time resolved fluorescence assay for testing cross-reactivity.
In this assay, labelled wild type mature human IL-6 is used at a
final concentration of the Kd of its interaction with the binding
member.
[0049] A binding member of the invention may cross-react with
cynomolgus IL-6. Cross-reactivity may be determined as inhibition
of labelled human IL-6 binding to the binding member immobilised on
a support, in the time resolved fluorescence assay described above.
For example, cynomolgus IL-6 may have an IC.sub.50 of less than 5
nM, e.g. less than 2.5 nM, e.g. about 1 nM, in this time resolved
fluorescence assay. Cynomolgus IL-6 may have an IC.sub.50 less than
10-fold different, e.g. less than 5-fold different, from the
IC.sub.50 of unlabeled human IL-6 in this assay.
[0050] A detailed protocol for the time resolved fluorescence assay
for determining cross-reactivity is provided in the Materials and
Methods section. Examples of cross-reactivity data obtained in this
assay are shown in Table 2 in Example 1.6.
[0051] As reported in Example 2.8, binding members described herein
showed high cross-reactivity with cynomolgus IL-6, and showed no or
limited cross-reactivity with rat, mouse or dog IL-6.
[0052] The cross-reactivity data indicate that the binding members
described herein bind an epitope on IL-6 that is conserved between
the human and cynomolgus IL-6 sequences, and is different in the
mouse, rat and dog IL-6 sequence compared with the human
sequence.
[0053] The binding members described herein are believed to bind
the "site 1" region of IL-6, which is the region that interacts
with IL-6R.alpha.. Binding members of the invention may thus
competitively inhibit IL-6 binding to IL-6R.alpha., thereby
neutralising biological effects of IL-6 that are mediated through
6R.alpha..
[0054] We investigated the ability of one of the antibodies
described herein, Antibody 18, to bind mutant human IL-6, in which
mutations were engineered in site 1 residues. As described in
Example 3, we identified mutations in human IL-6 that resulted in
reduced binding by Antibody 18, indicating that the mutated
residues were involved in recognition by Antibody 18 and may form
part of the epitope on IL-6 bound by this antibody.
[0055] For example, in a time resolved fluorescence assay for
inhibition of labelled wild type human IL-6 binding to Antibody 18
immobilised on a support, no inhibition was observed for Arg207Glu
mutant human IL-6 (SEQ ID NO: 177), indicating that Antibody 18
binds human IL-6 at residue Arg207.
[0056] Since Antibody 18 and Antibodies 2, 3, 4, 5, 7, 8, 10, 14,
16, 17, 19, 21, 22 and 23 were all derived from a parent antibody
CAN22C10, and all have structurally related CDRs, all these
antibody molecules are expected to bind the same or very similar
overlapping epitope. Accordingly, the epitope mapping results
obtained with Antibody 18 are also expected to be representative
for CAN22D10 the other optimised antibodies described herein.
[0057] A binding member of the invention may bind human IL-6 at
Phe102 and/or Ser204. A binding member of the invention may also
bind human IL-6 at Arg207. Optionally a binding member may bind
flanking residues or structurally neighbouring residues in the IL-6
molecule, in addition to binding Phe102 and/or Ser 204. By
convention, residue numbering corresponds to full length human IL-6
(SEQ ID NO: 161). However, binding may be determined using mature
human IL-6. Binding to IL-6 residues is as determined by site
directed mutagenesis, as explained below.
[0058] Mutagenesis of single amino acids and regions of proteins in
order to correlate structure with activity is well known to one
skilled in the art and has been used to define regions of proteins
that bind to antibodies [41]. Binding to and/or neutralisation of
mutant human IL-6 may be used to assess whether a binding member
binds Phe102, Ser204 and/or Arg207. Absence of binding or
neutralisation, or significantly reduced binding or neutralisation,
with mutant IL-6 compared with wild-type indicates that a binding
member binds the mutated residue.
[0059] Binding to a residue in IL-6 may be determined using IL-6
mutated at the selected residue in a time resolved fluorescence
assay of inhibition of labelled wild type human IL-6 binding to the
binding member immobilised on a support, wherein the labelled wild
type mature human IL-6 is at a final concentration equal to the Kd
of its interaction with the binding member. An example of this
assay and competition data obtained are shown in Example 3, with
results presented in Table 10. Where the mutant IL-6 does not
inhibit binding of labelled wild type IL-6 to the binding member,
or where the mutant IL-6 has an IC50 greater than that of unlabeled
wild type IL-6 (e.g. more than 10-fold or 100-fold greater), this
indicates that the mutated residue is bound by the binding member.
Phe102Glu mutant human IL-6 (SEQ ID NO: 175), Ser204Glu mutant
human IL-6 (SEQ ID NO: 176), and/or Arg207Glu mutant human IL-6
(SEQ ID NO: 177) may show no inhibition, or may have an IC.sub.50
more than 100 fold greater than the IC.sub.50 of wild type human
IL-6 (SEQ ID NO: 165), in a time resolved fluorescence assay for
inhibition of labelled wild type human IL-6 binding to a binding
member of the invention immobilised on a support, wherein the
labelled wild type human IL-6 is at a final concentration equal to
the Kd of its interaction with the binding member.
[0060] A binding member of the invention may optionally not bind
and/or neutralise mutant human IL-6 having a mutation at residue
Phe102, Ser204 and/or Arg207, e.g. mutation Phe102Glu, Ser204Glu,
Ser204Tyr and/or Arg207Glu. Examples of mutant human IL-6 sequences
are SEQ ID NOS: 175-177). Thus, a binding member of the invention
may not inhibit binding of one or more of these mutant IL-6
molecules to IL-6R.alpha..
[0061] A binding member of the invention may comprise an antibody
molecule, e.g. a human antibody molecule. The binding member
normally comprises an antibody VH and/or VL domain. VH and VL
domains of binding members are also provided as part of the
invention. Within each of the VH and VL domains are complementarity
determining regions, ("CDRs"), and framework regions, ("FRs"). A VH
domain comprises a set of HCDRs, and a VL domain comprises a set of
LCDRs. An antibody molecule may comprise an antibody VH domain
comprising a VH CDR1, CDR2 and CDR3 and a framework. It may
alternatively or also comprise an antibody VL domain comprising a
VL CDR1, CDR2 and CDR3 and a framework. A VH or VL domain framework
comprises four framework regions, FR1, FR2, FR3 and FR4,
interspersed with CDRs in the following structure:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0062] Examples of antibody VH and VL domains and CDRs according to
the present invention are as listed in the appended sequence
listing that forms part of the present disclosure. Further CDRs are
disclosed below and in Table 7. All VH and VL sequences, CDR
sequences, sets of CDRs and sets of HCDRs and sets of LCDRs
disclosed herein represent aspects and embodiments of the
invention. As described herein, a "set of CDRs" comprises CDR1,
CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and
HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless
otherwise stated, a "set of CDRs" includes HCDRs and LCDRs.
Typically binding members of the invention are monoclonal
antibodies.
[0063] A binding member of the invention may comprise an
antigen-binding site within a non-antibody molecule, normally
provided by one or more CDRs e.g. a set of CDRs in a non-antibody
protein scaffold, as discussed further below.
[0064] Described herein is a binding member comprising the parent
set of CDRs as shown in Table 7 for parent CAN022D10, in which
HCDR1 is SEQ ID NO: 3 (Kabat residues 31-35), HCDR2 is SEQ ID NO: 4
(Kabat residues 50-65), HCDR3 is SEQ ID NO: 5 (Kabat residues
95-102), LCDR1 is SEQ ID NO: 8 (Kabat residues 24-34), LCDR2 is SEQ
ID NO: 9 (Kabat residues 50-56) and LCDR3 is SEQ ID NO: 10 (Kabat
residues 89-97).
[0065] A binding member of the invention may comprise one or more
CDRs as described herein, e.g. a CDR3, and optionally also a CDR1
and CDR2 to form a set of CDRs. The CDR or set of CDRs may be a
parent CDR or parent set of CDRs, or may be a CDR or set of CDRs of
any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22
or 23, or may be a variant thereof as described herein.
[0066] For example, a binding member or a VL domain according to
the invention may comprise an LCDR3 having amino acid sequence SEQ
ID NO: 120.
[0067] A binding member may comprise a set of H and/or L CDRs of
the parent antibody or any of antibodies 2, 3, 4, 5, 7, 8, 10, 14,
16, 17, 18, 19, 21, 22 or 23 with one or more amino acid mutations
within the disclosed set of H and/or L CDRs. Amino acid mutations
are substitutions, deletions or insertions of one amino acid. For
example, there may be up to 20, e.g. up to 12, 11, 10, 9, 8, 7, 6,
5, 4, 3 or 2 mutations e.g. substitutions, within the set of H
and/or L CDRs. For example, there may be up to 6, 5, 4, 3 or 2
mutations, e.g. substitutions, in HCDR3 and/or there may be up to
6, 5, 4, 3, or 2 mutations, e.g. substitutions, in LCDR3. HCDR3
and/or LCDR3 may optionally contain an insertion or deletion of one
amino acid as compared with the disclosed set of H and/or LCDRs.
Substitutions may for example be at the positions substituted in
any of Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22
or 23, as shown in Table 7. Thus, substitutions may optionally be
at Kabat numbers selected from the following:
[0068] Kabat residue 35 in HCDR1;
[0069] Kabat residue 64 in HCDR2;
[0070] Kabat residue 96, 97, 98, 99, 100, 100A, 100B, 100C and/or
102 in HCDR3;
[0071] Kabat residue 34 in LCDR1;
[0072] Kabat residue 89, 90, 91, 92, 93, 94, 96 or 97 in LCDR3.
[0073] The amino acid mutations may comprise mutations as shown in
Table 7, e.g. amino acid substitutions as indicated.
[0074] For example, a binding member or a VH domain according to
the invention may comprise the parent HCDR1 with Kabat residue Ile
35 replaced by Thr or Val.
[0075] A binding member or a VH domain according to the invention
may comprise the parent HCDR2 with Kabat residue Lys 64 replaced by
Arg.
[0076] A binding member or a VH domain may comprise the parent
HCDR3 with one or more of the following mutations:
[0077] Kabat residue Ala 96 replaced by Glu;
[0078] Kabat residue Asp 97 replaced by Glu or Asn;
[0079] Kabat residue Asp 98 replaced by Gly, Glu or His;
[0080] Kabat residue His 99 replaced by Gly or Thr;
[0081] Kabat residue Tyr 100 replaced by Pro, Asn, Arg, Trp or
Ala;
[0082] Kabat residue Tyr 100A replaced by Ala, Arg, Thr, Gly, Asn,
Pro or Ser;
[0083] Kabat residue 100B replaced by His, Trp, Gln, Pro or
Thr;
[0084] Kabat residue Ile 100C replaced by Ala, Val, His, Tyr or
Leu;
[0085] Ile inserted at Kabat residue 100D;
[0086] Kabat residue Val 102 is replaced by Leu, His, Met or
Ile.
[0087] Thus, a binding member or a VH domain of the invention may
comprise an HCDR3 wherein Kabat residue 100D is Ile or wherein
Kabat residue 100D is absent.
[0088] A binding member or a VL domain of the invention may
comprise the parent LCDR1 in which Kabat residue Ala 34 is replaced
by Thr.
[0089] A binding member of a VL domain of the invention may
comprise the parent LCDR3 with one or more of the following
mutations:
[0090] Kabat residue Gln 89 replaced by Met or Ala;
[0091] Kabat residue Gln 90 replaced by Asn, Ser or Ala;
[0092] Kabat residue Ser 91 replaced by Asn, Gly, Ala or His;
[0093] Kabat residue Tyr 92 replaced by Trp, Ser, Lys or Phe;
[0094] Kabat residue Ser 93 replaced by Leu, Lys, Arg or Ala;
[0095] Kabat residue Thr 94 replaced by Ala, Gly or Pro;
[0096] Kabat residue Pro 95 deleted;
[0097] Kabat residue Trp 96 replaced by Gly;
[0098] Kabat residue Thr 97 replaced by Ser.
[0099] Thus, a binding member or a VL domain of the invention may
comprise an LCDR3 in which Kabat residue 95 is Pro or wherein Kabat
residue 95 is absent.
[0100] The invention provides an isolated binding member for human
IL-6 comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2
and LCDR3, wherein the set of CDRs has 22 or fewer amino acid
alterations, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4, 3, 2, 1 alterations or no alterations, from a
set of CDRs in which:
[0101] HCDR1 has amino acid sequence SEQ ID NO: 3;
[0102] HCDR2 has amino acid sequence SEQ ID NO: 4;
[0103] 10 HCDR3 has amino acid sequence SEQ ID NO: 115;
[0104] LCDR1 has amino acid sequence SEQ ID NO: 8;
[0105] LCDR2 has amino acid sequence SEQ ID NO: 9; and
[0106] LCDR3 has amino acid sequence SEQ ID NO: 120.
[0107] An amino acid alteration may be a substitution, insertion or
deletion. Examples of Kabat positions that may be substituted, and
examples of residue substitutions are discussed below, and Table 7
illustrates some of the substitutions.
[0108] As shown in Table 7, the length of HCDR3 and LCDR3 varied
between different optimised antibodies described herein. Relative
to the parent CDRs of CAN022D10, an insertion between Kabat
residues 100 to 102 (shown in Table 7 at Kabat residue 100D) was
observed in some antibodies, and a deletion between Kabat residues
92 to 97 was observed in other antibodies. The deletion at Kabat
residue 95 was not observed in combination with the insertion.
Thus, it may be advantageous for the longer, 12 residue HCDR3
sequences to be combined with the longer, 9 residue LCDR3
sequences, and it may be advantageous for the shorter, 11 residue
HCDR3 sequences to be combined with the shorter, 8 residue LCDR3
sequences.
[0109] According to the Kabat numbering system, residues of LCDR3
are numbered from 89 to 97. LCDR3 sequences shorter than 9 residues
are not envisaged by the Kabat numbering system. In the present
invention, binding members may have an LCDR3 shorter than 9
residues, e.g. LCDR3 may be 8 residues long, as shown in Table 7.
We number the 8 residues of LCDR389, 90, 91, 92, 93, 94, 96 and 97,
respectively. In Table 7, deletion is thus shown at Kabat residue
95. However, it will be appreciated that the effect of the deletion
is to reduce the length of the LCDR3 sequence, and that in
principle the deletion could be considered to be made at any of
residues 89 to 97, e.g. any of residues 92 to 97.
[0110] In HCDR3, the Kabat numbering system accommodates
variability in CDR length by extension of the numbering system
between Kabat residues 100 and 101, e.g. including residue 100A for
an HCDR3 of 9 residues, plus 100B for an HCDR3 of 10 residues, plus
100C for an HCDR3 of 11 residues, plus 100D for an HCDR3 of 12
residues, as appropriate. In Table 7, the insertion of an
additional amino acid in HCDR3 of some of the optimised clones
relative to the parent HCDR3 is shown at Kabat residue 100D.
However, it will be appreciated that in principle this insertion
may be considered to be made at any of Kabat residues 100 to
102.
[0111] As demonstrated herein, one or more insertions or deletions
may be present in one or more CDRs of a binding member, e.g. an
HCDR3 and/or LCDR3. For example, a binding member of the invention
may comprise a set of CDRs of any of Antibodies 2, 3, 4, 5, 7, 8,
10, 14, 16, 17, 18, 19, 21, 22 and 23, or a variant thereof as
described herein, wherein each CDR optionally has an insertion to
increase the length of the CDR by one residue or has a deletion of
one residue to decrease the length of the CDR by one residue.
Insertions and/or deletions may be made in HCDRs and/or an LCDRs,
e.g. in an HCDR3 and/or in an LCDR3.
[0112] For example, a binding member may for example comprise a set
of CDRs having 20 or fewer amino acid substitutions in a set of
CDRs wherein:
[0113] HCDR1 has amino acid sequence SEQ ID NO: 3;
[0114] HCDR2 has amino acid sequence SEQ ID NO: 4;
[0115] HCDR3 has amino acid sequence SEQ ID NO: 115;
[0116] LCDR1 has amino acid sequence SEQ ID NO: 8;
[0117] LCDR2 has amino acid sequence SEQ ID NO: 9; and
[0118] LCDR3 has amino acid sequence SEQ ID NO: 120;
[0119] wherein the binding member optionally has an insertion of
one residue to increase the length of the HCDR3 or a deletion of
one residue to decrease the length of the HCDR3, and/or
[0120] has an insertion of one residue to increase the length of
the LCDR3 or a deletion of one residue to decrease the length of
the LCDR3.
[0121] A binding member of the invention may have an insertion of
one residue in HCDR3 SEQ ID NO: 115 and/or an insertion of one
residue in LCDR3 SEQ ID NO: 120.
[0122] Insertions or deletions may be made at any point in the
CDRs. For example, in HCDR3 insertions or deletions may be of any
of Kabat residues 95-102, e.g. any of Kabat residues 100-102. For
example, in LCDR3 insertions or deletions may be of any of Kabat
residues 89 to 97, e.g. any of Kabat residues 92 to 97.
[0123] A binding member or VH domain of the invention may comprise
an HCDR1 in which Kabat residue 35 is Ile, Thr or Val.
[0124] A binding member or VH domain of the invention may comprise
an HCDR2 in which Kabat residue 64 is Lys or Arg.
[0125] A binding member or VH domain of the invention may comprise
an HCDR3 in which Kabat residue 95 is Trp and/or Kabat residue 101
is Asp.
[0126] A binding member or VH domain of the invention may comprise
an HCDR3 wherein:
[0127] Kabat residue 96 is Ala or Glu;
[0128] Kabat residue 97 is Asp, Glu or Asn;
[0129] Kabat residue 98 is Asp, Gly, Glu or His;
[0130] Kabat residue 99 is His, Gly or Thr;
[0131] Kabat residue 100 is Pro, Tyr, Asn, Arg, Trp or Ala;
[0132] Kabat residue 100A is Pro, Tyr, Ala, Arg, Thr, Gly, Asn, Pro
or Ser;
[0133] Kabat residue 100B is Trp, Tyr, His, Gln, Pro or Thr;
[0134] Kabat residue 100C is Ile, Ala, Val, His, Tyr or Leu;
and
[0135] Kabat residue 102 is Leu, Val, His, Met or Ile.
[0136] A binding member or VL domain of the invention may comprise
an LCDR1 in which Kabat residue 34 is Ala or Thr.
[0137] A binding member or VL domain of the invention may comprise
an LCDR3 wherein:
[0138] Kabat residue 89 is Gln, Met or Ala;
[0139] Kabat residue 90 is Gln, Asn, Ser or Ala;
[0140] Kabat residue 91 is Ser, Asn, Gly, Ala or His;
[0141] Kabat residue 92 is Trp, Tyr, Ser, Lys or Phe;
[0142] Kabat residue 93 is Leu, Ser, Lys, Arg or Ala;
[0143] Kabat residue 94 is Gly, Thr, Ala or Pro;
[0144] Kabat residue 96 is Gly or Trp; and
[0145] Kabat residue 97 is Ser or Thr.
[0146] The invention provides binding members comprising an HCDR1,
HCDR2 and/or HCDR3 of the parent or any of antibodies 2, 3, 4, 5,
7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23, and/or an LCDR1, LCDR2
and/or LCDR3 of the parent or any of antibodies 2, 3, 4, 5, 7, 8,
10, 14, 16, 17, 18, 19, 21, 22 and 23 e.g. a set of CDRs of the
parent or any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18,
19, 21, 22 and 23 shown in Table 7.
[0147] For example, a binding member of the invention may comprise
a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3,
wherein: HCDR1 is SEQ ID NO: 113; HCDR2 is SEQ ID NO: 114; HCDR3 is
SEQ ID NO: 115; LCDR1 is SEQ ID NO: 118; LCDR2 is SEQ ID NO: 119;
and LCDR3 is SEQ ID NO: 120, representing the CDRs of Antibody
18.
[0148] The binding member may comprise a set of VH CDRs of one of
these antibodies.
[0149] Optionally it may also comprise a set of VL CDRs of one of
these antibodies, and the VL CDRs may be from the same or a
different antibody as the VH CDRs.
[0150] A VH domain comprising a set of HCDRs of the parent or any
of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and
23, and/or a VL domain comprising a set of LCDRs of the parent or
any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22
and 23 are also provided by the invention.
[0151] Typically, a VH domain is paired with a VL domain to provide
an antibody antigen-binding site, although as discussed further
below a VH or VL domain alone may be used to bind antigen. The
antibody 2 VH domain may be paired with the antibody 2 VL domain,
so that an antibody antigen-binding site is formed comprising both
the antibody 2 VH and VL domains. Analogous embodiments are
provided for the other VH and VL domains disclosed herein. In other
embodiments, the antibody 2 VH is paired with a VL domain other
than the antibody VL. Light-chain promiscuity is well established
in the art. Again, analogous embodiments are provided by the
invention for the other VH and VL domains disclosed herein.
[0152] Thus, the VH of the parent or of any of antibodies 2, 3, 4,
5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23 may be paired with
the VL of the parent or of any of antibodies 2, 3, 4, 5, 7, 8, 10,
14, 16, 17, 18, 19, 21, 22 and 23.
[0153] A binding member may comprise an antibody molecule having
one or more CDRs, e.g. a set of CDRs, within an antibody framework.
For example, one or more CDRs or a set of CDRs of an antibody may
be grafted into a framework (e.g. human framework) to provide an
antibody molecule. The framework regions may be of human germline
gene segment sequences. Thus, the framework may be germlined,
whereby one or more residues within the framework are changed to
match the residues at the equivalent position in the most similar
human germline framework. The skilled person can select a germline
segment that is closest in sequence to the framework sequence of
the antibody before germlining and test the affinity or activity of
the antibodies to confirm that germlining does not significantly
reduce antigen binding or potency in assays described herein. Human
germline gene segment sequences are known to those skilled in the
art and can be accessed for example from the VBase compilation.
[0154] A binding member of the invention may be an isolated human
antibody molecule having a VH domain comprising a set of HCDRs in a
human germline framework, e.g. Vh3_DP-86_(3-66). Thus, the VH
domain framework regions FR1, FR2 and/or FR3 may comprise framework
regions of human germline gene segment Vh3_DP-86_(3-66) and/or may
be germlined by mutating framework residues to match the framework
residues of this human germline gene segment. FR4 may comprise a
framework region of human germline j segment JH2. The amino acid
sequence of VH FR1 may be SEQ ID NO: 167. The amino acid sequence
of VH FR2 may be SEQ ID NO: 168. The amino acid sequence of VH FR3
may be SEQ ID NO: 169. The amino acid sequence of VH FR4 may be SEQ
ID NO: 170.
[0155] Normally the binding member also has a VL domain comprising
a set of LCDRs, e.g. in a human germline framework, e.g. Vkl L12.
Thus, the VL domain framework regions may comprise framework
regions FR1, FR2 and/or FR3 of human germline gene segment Vkl L12
and/or may be germlined by mutating framework residues to match the
framework residues of this human germline gene segment. FR4 may
comprise a framework region of human germline j segment JK2. The
amino acid sequence of VL FR1 may be SEQ ID NO: 171. The amino acid
sequence of VL FR2 may be SEQ ID NO: 172. The amino acid sequence
of VL FR3 may be SEQ ID NO: 173. The amino acid sequence of VL FR4
may be SEQ ID NO: 174.
[0156] A germlined VL domain may or may not be germlined at the
Vernier residue or residues, but is normally not.
[0157] An antibody molecule or a VH domain of the invention may
comprise the following set of heavy chain framework regions:
[0158] FR1 SEQ ID NO: 167;
[0159] FR2 SEQ ID NO: 168;
[0160] FR3 SEQ ID NO: 169;
[0161] FR4 SEQ ID NO: 170;
[0162] or may comprise the said set of heavy chain framework
regions with one, two, three, four or five amino acid alterations,
e.g. substitutions.
[0163] An antibody molecule or a VL domain of the invention may
comprise the following set of light chain framework regions:
[0164] FR1 SEQ ID NO: 171;
[0165] FR2 SEQ ID NO: 172;
[0166] FR3 SEQ ID NO: 173;
[0167] FR4 SEQ ID NO: 174;
[0168] or may comprise the said set of light chain framework
regions with one, two, three, four or five amino acid alterations,
e.g. substitutions.
[0169] An amino acid alteration may be a substitution, an insertion
or a deletion.
[0170] For example, an antibody molecule of the invention may
comprise a set of heavy and light chain framework regions, wherein
heavy chain FR1 is SEQ ID NO: 167;
[0171] heavy chain FR2 is SEQ ID NO: 168;
[0172] heavy chain FR3 is SEQ ID NO: 169;
[0173] heavy chain FR4 is SEQ ID NO: 170;
[0174] light chain FR1 is SEQ ID NO: 171;
[0175] light chain FR2 is SEQ ID NO: 172;
[0176] light chain FR3 is SEQ ID NO: 173;
[0177] light chain FR4 is SEQ ID NO: 174;
[0178] or may comprise the said set of heavy and light chain
framework regions with 10 or fewer, e.g. five or fewer, amino acid
alterations, e.g. substitutions. For example there may be one or
two amino acid substitutions in the said set of heavy and light
chain framework regions.
[0179] A non-germlined antibody molecule has the same CDRs, but
different frameworks, compared with a germlined antibody molecule.
Of the antibody sequences shown herein in the appended sequence
listing, sequences of antibody nos 7, 10, 17 and 18 are germlined.
Germlined antibodies 2 to 5, 8, 14, 16, 19 and 21 to 23 may be
produced by germlining framework regions of the VH and VL domain
sequences shown herein for these antibodies.
[0180] The 3' cgt codon, and corresponding Arginine residue, shown
in the nucleotide and amino acid sequences for the kappa VL domains
of Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and
23 respectively were included in the expressed scFv and IgG
sequences of these antibodies. The C terminal Arginine residue of
the sequences corresponds to Kabat residue 108. The origin of this
residue and its encoding triplet cgt is explained below.
[0181] To express the light chain of the IgG, a nucleotide sequence
encoding the antibody light chain was provided, comprising a first
exon encoding the VL domain, a second exon encoding the CL domain,
and an intron separating the first exon and the second exon. Under
normal circumstances, the intron is spliced out by cellular mRNA
processing machinery, joining the 3' end of the first exon to the
5' end of the second exon. Thus, when DNA having the said
nucleotide sequence was expressed as RNA, the first and second
exons were spliced together. Translation of the spliced RNA
produces a polypeptide comprising the VL domain and CL domain.
[0182] The choice of constant domain is significant in that for
kappa light chains the bridging amino acid is arginine, formed by
the cga codon, where the first cytosine is encoded in exon 1 and
the guanine and adenine are encoded in exon 2.
[0183] After splicing, for Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16,
17, 18, 19, 21, 22 and 23, the Arg at Kabat residue 108 is encoded
by the last base (c) of the VL domain framework 4 sequence and the
first two bases (gt) of the CL domain.
[0184] The Arginine residue at Kabat residue 108 may be considered
to be the C terminal residue of the VL domain of the antibody
molecule.
[0185] A binding member of the invention may be one which competes
for binding to IL-6 with any binding member that (i) binds IL-6 and
(ii) comprises a binding member, VH and/or VL domain, CDR e.g.
HCDR3, and/or set of CDRs disclosed herein.
[0186] Competition between binding members may be assayed easily in
vitro, for example using ELISA and/or by tagging a specific
reporter molecule to one binding member which can be detected in
the presence of one or more other untagged binding members, to
enable identification of binding members which bind the same
epitope or an overlapping epitope. Such methods are readily known
to one of ordinary skill in the art, and are described in more
detail herein (see the Detailed Description, and the epitope
competition assays in the Materials and Methods section of the
Examples.) Thus, a further aspect of the present invention provides
a binding member comprising a human antibody antigen-binding site
that competes with an antibody molecule, for example an antibody
molecule comprising a VH and/or VL domain, CDR e.g. HCDR3 or set of
CDRs of the parent antibody or any of antibodies 2, 3, 4, 5, 7, 8,
10, 14, 16, 17, 18, 19, 21, 22 and 23, for binding to IL-6.
[0187] In further aspects, the invention provides an isolated
nucleic acid which comprises a sequence encoding a binding member,
VH domain and/or VL domain according to the present invention, and
methods of preparing a binding member, a VH domain and/or a VL
domain of the invention, which comprise expressing said nucleic
acid under conditions to bring about production of said binding
member, VH domain and/or VL domain, and recovering it.
[0188] Another aspect of the present invention provides nucleic
acid, generally isolated, encoding a VH CDR or VL CDR sequence
disclosed herein.
[0189] A further aspect provides a host cell containing or
transformed with nucleic acid of the invention.
[0190] Further aspects of the present invention provide for
compositions containing binding members of the invention, and their
use in methods of binding, inhibiting and/or neutralising IL-6,
including methods of treatment of the human or animal body by
therapy.
[0191] Binding members according to the invention may be used in a
method of treatment or diagnosis, such as a method of treatment
(which may include prophylactic treatment) of a disease or disorder
in the human or animal body (e.g. in a human patient), which
comprises administering to said patient an effective amount of a
binding member of the invention. Conditions treatable in accordance
with the present invention include any in which IL-6 plays a role,
as discussed in detail elsewhere herein.
[0192] These and other aspects of the invention are described in
further detail below.
Terminology
[0193] It is convenient to point out here that "and/or" where used
herein is to be taken as specific disclosure of each of the two
specified features or components with or without the other. For
example "A and/or B" is to be taken as specific disclosure of each
of (i) A, (ii) B and (iii) A and B, just as if each is set out
individually herein.
[0194] 5 IL-6 and IL-6 Receptor
[0195] IL-6 is interleukin 6. IL-6 may also be referred to herein
as "the antigen".
[0196] The full length amino acid sequence of human IL-6 is SEQ ID
NO: 161. This sequence is cleaved in vivo to remove an N-terminal
leader peptide, to produce mature IL-6. Mature human IL-6 has amino
acid sequence SEQ ID NO: 165. The mature sequence represents the in
vivo circulating IL-6, which is the target antigen for therapeutic
and in vivo diagnostic applications as described herein.
Accordingly, IL-6 referred to herein is normally mature human IL-6,
unless otherwise indicated by context.
[0197] IL-6 may be conjugated to a detectable label, such as HIS
FLAG, e.g. for use in assays as described herein. For example, a
fusion protein comprising IL-6 conjugated to a HIS FLAG sequence
may be used. A sequence of HIS FLAG tagged human IL-6 is SEQ ID NO:
162.
[0198] IL-6 receptor a, IL-6Ra, is the receptor for interleukin 6.
IL-6Ra is also known as IL-6R.alpha., IL-6Ra, IL-6R and CD126.
IL-6Ra exists in vivo in a transmembrane form and in a soluble
form. References to IL-6Ra may be transmembrane IL-6Ra and/or
soluble IL-6Ra unless otherwise indicated by context.
[0199] IL-6 receptor referred to herein is normally human IL-6
receptor, unless otherwise indicated. An amino acid sequence of
human soluble IL-6Ra (sIL-6Ra, sIL-6R) is SEQ ID NO: 163. An amino
acid sequence of human transmembrane IL-6Ra is SEQ ID NO: 164.
[0200] IL-6 binds IL-6Ra to form a complex, IL-6:IL-6Ra. The
complex may be either soluble (with sIL-6Ra) or membrane bound
(with transmembrane IL-6Ra). When the IL-6Ra is the soluble form,
the complex is designated IL-6:sIL-6Ra. References to IL-6:IL-6Ra
may include IL-6 complexed with transmembrane IL-6Ra or with
soluble IL-6Ra, unless otherwise indicated by context.
gp130
[0201] gp130 is a receptor for the IL-6:IL-6Ra complex. Cloning and
characterisation of gp130 is reported in Hibi et al, Cell 63:1149
1157 (1990). A sequence of human gp130 is set out in SEQ ID NO:
166.
Binding Member
[0202] This describes one member of a pair of molecules that bind
one another. The members of a binding pair may be naturally derived
or wholly or partially synthetically produced. One member of the
pair of molecules has an area on its surface, or a cavity, which
binds to and is therefore complementary to a particular spatial and
polar organization of the other member of the pair of molecules.
Examples of types of binding pairs are antigen-antibody,
biotin-avidin, hormone-hormone receptor, receptor-ligand,
enzyme-substrate. The present invention is concerned with
antigen-antibody type reactions.
[0203] A binding member normally comprises a molecule having an
antigen-binding site. For example, a binding member may be an
antibody molecule or a non-antibody protein that comprises an
antigen-binding site.
[0204] An antigen binding site may be provided by means of
arrangement of CDRs on non-antibody protein scaffolds, such as
fibronectin or cytochrome B etc. [42, 43, 44], or by randomising or
mutating amino acid residues of a loop within a protein scaffold to
confer binding specificity for a desired target. Scaffolds for
engineering novel binding sites in proteins have been reviewed in
detail by Nygren et al. [44]. Protein scaffolds for antibody mimics
are disclosed in WO/0034784, which is herein incorporated by
reference in its entirety, in which the inventors describe proteins
(antibody mimics) that include a fibronectin type III domain having
at least one randomised loop. A suitable scaffold into which to
graft one or more CDRs, e.g. a set of HCDRs, may be provided by any
domain member of the immunoglobulin gene superfamily. The scaffold
may be a human or non-human protein. An advantage of a non-antibody
protein scaffold is that it may provide an antigen-binding site in
a scaffold molecule that is smaller and/or easier to manufacture
than at least some antibody molecules. Small size of a binding
member may confer useful physiological properties, such as an
ability to enter cells, penetrate deep into tissues or reach
targets within other structures, or to bind within protein cavities
of the target antigen. Use of antigen binding sites in non-antibody
protein scaffolds is reviewed in Wess, 2004 [45]. Typical are
proteins having a stable backbone and one or more variable loops,
in which the amino acid sequence of the loop or loops is
specifically or randomly mutated to create an antigen-binding site
that binds the target antigen. Such proteins include the
IgG-binding domains of protein A from S. aureus, transferrin,
tetranectin, fibronectin (e.g. 10th fibronectin type III domain),
lipocalins as well as gamma-crystalline and other Affilin.TM.
scaffolds (Scil Proteins). Examples of other approaches include
synthetic "Microbodies" based on cyclotides--small proteins having
intra-molecular disulphide bonds, Microproteins (Versabodies.TM.,
Amunix) and ankyrin repeat proteins (DARPins, Molecular
Partners).
[0205] In addition to antibody sequences and/or an antigen-binding
site, a binding member according to the present invention may
comprise other amino acids, e.g. forming a peptide or polypeptide,
such as a folded domain, or to impart to the molecule another
functional characteristic in addition to ability to bind antigen.
Binding members of the invention may carry a detectable label, or
may be conjugated to a toxin or a targeting moiety or enzyme (e.g.
via a peptidyl bond or linker). For example, a binding member may
comprise a catalytic site (e.g. in an enzyme domain) as well as an
antigen binding site, wherein the antigen binding site binds to the
antigen and thus targets the catalytic site to the antigen. The
catalytic site may inhibit biological function of the antigen, e.g
by cleavage.
[0206] Although, as noted, CDRs can be carried by non-antibody
scaffolds, the structure for carrying a CDR or a set of CDRs of the
invention will generally be an antibody heavy or light chain
sequence or substantial portion thereof in which the CDR or set of
CDRs is located at a location corresponding to the CDR or set of
CDRs of naturally occurring VH and VL antibody variable domains
encoded by rearranged immunoglobulin genes. The structures and
locations of immunoglobulin variable domains may be determined by
reference to Kabat, et al., 1987 [46], and updates thereof. A
number of academic and commercial on-line resources are available
to query this database. For example, see ref. [47] and the
associated on line resource, currently at the web address of
http://www.bioinf.org.uk/abs/simkab.html.
[0207] By CDR region or CDR, it is intended to indicate the
hypervariable regions of the heavy and light chains of the
immunoglobulin as defined by Kabat et al. 1991 [48], and later
editions. An antibody typically contains 3 heavy chain CDRs and 3
light chain CDRs. The term CDR or CDRs is used here in order to
indicate, according to the case, one of these regions or several,
or even the whole, of these regions which contain the majority of
the amino acid residues responsible for the binding by affinity of
the antibody for the antigen or the epitope which it
recognizes.
[0208] Among the six short CDR sequences, the third CDR of the
heavy chain (HCDR3) has a greater size variability (greater
diversity essentially due to the mechanisms of arrangement of the
genes which give rise to it). It may be as short as 2 amino acids
although the longest size known is 26. CDR length may also vary
according to the length that can be accommodated by the particular
underlying framework. Functionally, HCDR3 plays a role in part in
the determination of the specificity of the antibody [refs. 49, 50,
51, 52, 53, 54, 55, 56].
[0209] HCDR1 may be 5 amino acids long, consisting of Kabat
residues 31-35.
[0210] HCDR2 may be 17 amino acids long, consisting of Kabat
residues 50-65.
[0211] HCDR3 may be 11 or 12 amino acids long, consisting of Kabat
residues 95-102, optionally including Kabat residue 100D.
[0212] LCDR1 may be 11 amino acids long, consisting of Kabat
residues 24-34.
[0213] LCDR2 may be 7 amino acids long, consisting of Kabat
residues 50-56.
[0214] LCDR3 may be 8 or 9 amino acids long, consisting of Kabat
residues 89-97, optionally including Kabat residue 95.
Antibody Molecule
[0215] This describes an immunoglobulin whether natural or partly
or wholly synthetically produced. The term also covers any
polypeptide or protein comprising an antibody antigen-binding site.
It must be understood here that the invention does not relate to
the antibodies in natural form, that is to say they are not in
their natural environment but that they have been able to be
isolated or obtained by purification from natural sources, or else
obtained by genetic recombination, or by chemical synthesis, and
that they can then contain unnatural amino acids as will be
described later. Antibody fragments that comprise an antibody
antigen-binding site include, but are not limited to, molecules
such as Fab, Fab', Fab'-SH, scFv, Fv, dAb and Fd. Various other
antibody molecules including one or more antibody antigen-binding
sites have been engineered, including for example Fab2, Fab3,
diabodies, triabodies, tetrabodies and minibodies. Antibody
molecules and methods for their construction and use are described
in [57].
[0216] It is possible to take monoclonal and other antibodies and
use techniques of recombinant DNA technology to produce other
antibodies or chimeric molecules that bind the target antigen. Such
techniques may involve introducing DNA encoding the immunoglobulin
variable region, or the CDRs, of an antibody to the constant
regions, or constant regions plus framework regions, of a different
immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or
EP-A-239400, and a large body of subsequent literature. A hybridoma
or other cell producing an antibody may be subject to genetic
mutation or other changes, which may or may not alter the binding
specificity of antibodies produced.
[0217] As antibodies can be modified in a number of ways, the term
"antibody molecule" should be construed as covering any binding
member or substance having an antibody antigen-binding site with
the required specificity and/or binding to antigen. Thus, this term
covers antibody fragments and derivatives, including any
polypeptide comprising an antibody antigen-binding site, whether
natural or wholly or partially synthetic. Chimeric molecules
comprising an antibody antigen-binding site, or equivalent, fused
to another polypeptide (e.g. derived from another species or
belonging to another antibody class or subclass) are therefore
included. Cloning and expression of chimeric antibodies are
described in EP-A-0120694 and EP-A-0125023, and a large body of
subsequent literature.
[0218] Further techniques available in the art of antibody
engineering have made it possible to isolate human and humanised
antibodies. For example, human hybridomas can be made as described
by Kontermann & Dubel [58]. Phage display, another established
technique for generating binding members has been described in
detail in many publications, such as Kontermann & Dubel [58]
and WO92/01047 (discussed further below), and US patents U.S. Pat.
Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907,
5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447,
6,291,650, 6,492,160, 6,521,404. Transgenic mice in which the mouse
antibody genes are inactivated and functionally replaced with human
antibody genes while leaving intact other components of the mouse
immune system, can be used for isolating human antibodies [59].
Humanised antibodies can be produced using techniques known in the
art such as those disclosed in for example WO91/09967, U.S. Pat.
No. 5,585,089, E9592106, U.S. Pat. Nos. 565,332 and WO93/17105.
Further, WO2004/006955 describes methods for humanising antibodies,
based on selecting variable region framework sequences from human
antibody genes by comparing canonical CDR structure types for CDR
sequences of the variable region of a non-human antibody to
canonical CDR structure types for corresponding CDRs from a library
of human antibody sequences, e.g. germline antibody gene segments.
Human antibody variable regions having similar canonical CDR
structure types to the non-human CDRs torm a subset of member human
antibody sequences from which to select human framework sequences.
The subset members may be further ranked by amino acid similarity
between the human and the non-human CDR sequences. In the method of
WO2004/006955, top ranking human sequences are selected to provide
the framework sequences for constructing a chimeric antibody that
functionally replaces human CDR sequences with the non-human CDR
counterparts using the selected subset member human frameworks,
thereby providing a humanized antibody of high affinity and low
immunogenicity without need for comparing framework sequences
between the non-human and human antibodies. Chimeric antibodies
made according to the method are also disclosed.
[0219] Synthetic antibody molecules may be created by expression
from genes generated by means of oligonucleotides synthesized and
assembled within suitable expression vectors, for example as
described by Knappik et al. [60] or Krebs et al. [61].
[0220] It has been shown that fragments of a whole antibody can
perform the function of binding antigens. Examples of binding
fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1
domains; (ii) the Fd fragment consisting of the VH and CH1 domains;
(iii) the Fv fragment consisting of the VL and VH domains of a
single antibody; (iv) the dAb fragment [62, 63, 64], which consists
of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2
fragments, a bivalent fragment comprising two linked Fab fragments
(vii) single chain Fv molecules (scFv), wherein a VH domain and a
VL domain are linked by a peptide linker which allows the two
domains to associate to form an antigen binding site [65, 66];
(viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix)
"diabodies", multivalent or multispecific fragments constructed by
gene fusion (WO94/13804; [67]). Fv, scFv or diabody molecules may
be stabilized by the incorporation of disulphide bridges linking
the VH and VL domains [68]. Minibodies comprising a scFv joined to
a CH3 domain may also be made [69]. Other examples of binding
fragments are Fab', which differs from Fab fragments by the
addition of a few residues at the carboxyl terminus of the heavy
chain CH1 domain, including one or more cysteines from the antibody
hinge region, and Fab'-SH, which is a Fab' fragment in which the
cysteine residue(s) of the constant domains bear a free thiol
group.
[0221] Qui et al. [70] described antibody molecules containing just
two CDRs linked by a framework region. CDR3 from the VH or VL
domain was linked to the CDR1 or CDR2 loop of the other domain.
Linkage was through the C terminus of the selected CDR1 or CDR2 to
the N terminus of the CDR3, via a FR region. Qui et al. selected
the FR region having the fewest hydrophobic patches. The best
combination for the antibody tested was found to be VL CDR1 linked
by VH FR2 to VH CDR3 (VHCDR1-VHFR2-VLCDR3). At a molecular weight
of around 3 kDa, these antibody molecules offer advantages in terms
of improved tissue penetration as compared with full
immunoglobulins (approx. 150 kDa) or scFv (approx. 28 kDa).
[0222] Antibody fragments of the invention can be obtained starting
from a parent antibody molecule or any of the antibody molecules 2,
3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23, by methods
such as digestion by enzymes e.g. pepsin or papain and/or by
cleavage of the disulfide bridges by chemical reduction. In another
manner, the antibody fragments comprised in the present invention
can be obtained by techniques of genetic recombination likewise
well known to the person skilled in the art or else by peptide
synthesis by means of, for example, automatic peptide synthesizers,
such as those supplied by the company Applied Biosystems, etc., or
by nucleic acid synthesis and expression.
[0223] Functional antibody fragments according to the present
invention include any functional fragment whose half-life is
increased by a chemical modification, especially by PEGylation, or
by incorporation in a liposome.
[0224] A dAb (domain antibody) is a small monomeric antigen-binding
fragment of an antibody, namely the variable region of an antibody
heavy or light chain [64]. VH dAbs occur naturally in camelids
(e.g. camel, llama) and may be produced by immunizing a camelid
with a target antigen, isolating antigen-specific B cells and
directly cloning dAb genes from individual B cells. dAbs are also
producible in cell culture. Their small size, good solubility and
temperature stability makes them particularly physiologically
useful and suitable for selection and affinity maturation. Camelid
VH dAbs are being developed for therapeutic use under the name
"Nanobodies.TM.". A binding member of the present invention may be
a dAb comprising a VH or VL domain substantially as set out herein,
or a VH or VL domain comprising a set of CDRs substantially as set
out herein.
[0225] Bispecific or bifunctional antibodies form a second
generation of monoclonal antibodies in which two different variable
regions are combined in the same molecule [71]. Their use has been
demonstrated both in the diagnostic field and in the therapy field
from their capacity to recruit new effector functions or to target
several molecules on the surface of tumour cells. Where bispecific
antibodies are to be used, these may be conventional bispecific
antibodies, which can be manufactured in a variety of ways [72],
e.g. prepared chemically or from hybrid hybridomas, or may be any
of the bispecific antibody fragments mentioned above. These
antibodies can be obtained by chemical methods [73, 74] or somatic
methods [75, 76] but likewise and preferentially by genetic
engineering techniques which allow the heterodimerization to be
forced and thus facilitate the process of purification of the
antibody sought [77]. Examples of bispecific antibodies include
those of the BiTE' technology in which the binding domains of two
antibodies with different specificity can be used and directly
linked via short flexible peptides. This combines two antibodies on
a short single polypeptide chain. Diabodies and scFv can be
constructed without an Fc region, using only variable domains,
potentially reducing the effects of anti-idiotypic reaction.
[0226] Bispecific antibodies can be constructed as entire IgG, as
bispecific Fab'2, as Fab'PEG, as diabodies or else as bispecific
scFv. Further, two bispecific antibodies can be linked using
routine methods known in the art to form tetravalent antibodies.
Bispecific diabodies, as opposed to bispecific whole antibodies,
may also be particularly useful because they can be readily
constructed and expressed in E. coli. Diabodies (and many other
polypeptides, such as antibody fragments) of appropriate binding
specificities can be readily selected using phage display
(WO94/13804) from libraries. If one arm of the diabody is to be
kept constant, for instance, with a specificity directed against
IL-6, then a library can be made where the other arm is varied and
an antibody of appropriate specificity selected. Bispecific whole
antibodies may be made by alternative engineering methods as
described in Ridgeway et al., 1996 [78].
[0227] Various methods are available in the art for obtaining
antibodies against IL-6. The antibodies may be monoclonal
antibodies, especially of human, murine, chimeric or humanized
origin, which can be obtained according to the standard methods
well known to the person skilled in the art.
[0228] In general, for the preparation of monoclonal antibodies or
their functional fragments, especially of murine origin, it is
possible to refer to techniques which are described in particular
in the manual "Antibodies" [79] or to the technique of preparation
from hybridomas described by Kohler and Milstein [80].
[0229] Monoclonal antibodies can be obtained, for example, from the
B cells of an animal immunized against IL-6, or one of its
fragments containing the epitope recognized by said monoclonal
antibodies. Suitable fragments and peptides or polypeptides
comprising them are described herein, and may be used to immunise
animals to generate antibodies against IL-6. Said IL-6, or one of
its fragments, can especially be produced according to the usual
working methods, by genetic recombination starting with a nucleic
acid sequence contained in the cDNA sequence coding for IL-6 or
fragment thereof, by peptide synthesis starting from a sequence of
amino acids comprised in the peptide sequence of the IL-6 and/or
fragment thereof.
[0230] The monoclonal antibodies can, for example, be purified on
an affinity column on which IL-6 or one of its fragments containing
the epitope recognized by said monoclonal antibodies, has
previously been immobilized. More particularly, the monoclonal
antibodies can be purified by chromatography on protein A and/or G,
followed or not followed by ion-exchange chromatography aimed at
eliminating the residual protein contaminants as well as the DNA
and the LPS, in itself, followed or not followed by exclusion
chromatography on Sepharose gel in order to eliminate the potential
aggregates due to the presence of dimers or of other multimers. In
one embodiment, the whole of these techniques can be used
simultaneously or successively.
Antigen-Binding Site
[0231] This describes the part of a molecule that binds to and is
complementary to all or part of the target antigen. In an antibody
molecule it is referred to as the antibody antigen-binding site,
and comprises the part of the antibody that binds to and is
complementary to all or part of the target antigen. Where an
antigen is large, an antibody may only bind to a particular part of
the antigen, which part is termed an epitope. An antibody
antigen-binding site may be provided by one or more antibody
variable domains. An antibody antigen-binding site may comprise an
antibody light chain variable region (VL) and an antibody heavy
chain variable region (VH).
[0232] WO2006/072620 describes engineering of antigen binding sites
in structural (non-CDR) loops extending between beta strands of
immunoglobulin domains. An antigen binding site may be engineered
in a region of an antibody molecule separate from the natural
location of the CDRs, e.g. in a framework region of a VH or VL
domain, or in an antibody constant domain e.g. CH1 and/or CH3. An
antigen binding site engineered in a structural region may be
additional to, or instead of, an antigen binding site formed by
sets of CDRs of a VH and VL domain. Where multiple antigen binding
sites are present in an antibody molecule, they may bind the same
antigen (IL-6), thereby increasing valency of the binding member.
Alternatively, multiple antigen binding sites may bind different
antigens (IL-6 and one or more another antigen), and this may be
used to add effector functions, prolong half-life or improve in
vivo delivery of the antibody molecule.
Isolated
[0233] This refers to the state in which binding members of the
invention, or nucleic acid encoding such binding members, will
generally be in accordance with the present invention. Thus,
binding members, VH and/or VL domains, and encoding nucleic acid
molecules and vectors according to the present invention may be
provided isolated and/or purified, e.g. from their natural
environment, in substantially pure or homogeneous form, or, in the
case of nucleic acid, free or substantially free of nucleic acid or
genes of origin other than the sequence encoding a polypeptide with
the required function. Isolated members and isolated nucleic acid
will be free or substantially free of material with which they are
naturally associated, such as other polypeptides or nucleic acids
with which they are found in their natural environment, or the
environment in which they are prepared (e.g. cell culture) when
such preparation is by recombinant DNA technology practised in
vitro or in vivo. Members and nucleic acid may be formulated with
diluents or adjuvants and still for practical purposes be
isolated--for example the members will normally be mixed with
gelatin or other carriers if used to coat microtitre plates for use
in immunoassays, or will be mixed with pharmaceutically acceptable
carriers or diluents when used in diagnosis or therapy. Binding
members may be glycosylated, either naturally or by systems of
heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC 85110503)
cells, or they may be (for example if produced by expression in a
prokaryotic cell) unglycosylated.
[0234] Heterogeneous preparations comprising anti-IL-6 antibody
molecules also form part of the invention. For example, such
preparations may be mixtures of antibodies with full-length heavy
chains and heavy chains lacking the C-terminal lysine, with various
degrees of glycosylation and/or with derivatized amino acids, such
as cyclisation of an N-terminal glutamic acid to form a
pyroglutamic acid residue.
[0235] As used herein, the phrase "substantially as set out" refers
to the characteristic(s) of the relevant CDRs of the VH or VL
domain of binding members described herein will be either identical
or highly similar to the specified regions of which the sequence is
set out herein. As described herein, the phrase "highly similar"
with respect to specified region(s) of one or more variable
domains, it is contemplated that from 1 to about 5, e.g. from 1 to
4, including 1 to 3, or 1 or 2, or 3 or 4, amino acid substitutions
may be made in the CDR and/or VH or VL domain.
BRIEF DESCRIPTION OF THE FIGURE
[0236] FIG. 1. This FIGURE shows the effect of administration of an
anti-IL-6 antibody (Antibody 18) on human recombinant IL-6 induced
haptoglobin increase in the mouse in vivo.
DETAILED DESCRIPTION OF THE INVENTION
[0237] As noted above, a binding member in accordance with the
present invention modulates and may neutralise a biological
activity of IL-6. As described herein, IL-6-binding members of the
present invention may be optimised for neutralizing potency.
Generally, potency optimisation involves mutating the sequence of a
selected binding member (normally the variable domain sequence of
an antibody) to generate a library of binding members, which are
then assayed for potency and the more potent binding members are
selected. Thus selected "potency-optimised" binding members tend to
have a higher potency than the binding member from which the
library was generated. Nevertheless, high potency binding members
may also be obtained without optimisation, for example a high
potency binding member may be obtained directly from an initial
screen e.g. a biochemical neutralization assay. A "potency
optimized" binding member refers to a binding member with an
optimized potency of neutralization of a particular activity or
downstream function of IL-6. Assays and potencies are described in
more detail elsewhere herein. The present invention provides both
potency-optimized and non-optimized binding members, as well as
methods for potency optimization from a selected binding member.
The present invention thus allows the skilled person to generate
binding members having high potency.
[0238] In a further aspect, the present invention provides a method
of obtaining one or more binding members able to bind the antigen,
the method including bringing into contact a library of binding
members according to the invention and said antigen, and selecting
one or more binding members of the library able to bind said
antigen.
[0239] The library may be displayed on particles or molecular
complexes, e.g. replicable genetic packages, such as yeast,
bacterial or bacteriophage (e.g. T7) particles, viruses, cells or
covalent, ribosomal or other in vitro display systems, each
particle or molecular complex containing nucleic acid encoding the
antibody VH variable domain displayed on it, and optionally also a
displayed VL domain if present. Phage display is described in
WO92/01047 and e.g. US patents U.S. Pat. Nos. 5,969,108, 5,565,332,
5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793, 5,962,255,
6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and
6,521,404, each of which is herein incorporated by reference in
their entirety.
[0240] Following selection of binding members able to bind the
antigen and displayed on bacteriophage or other library particles
or molecular complexes, nucleic acid may be taken from a
bacteriophage or other particle or molecular complex displaying a
said selected binding member. Such nucleic acid may be used in
subsequent production of a binding member or an antibody VH or VL
variable domain by expression from nucleic acid with the sequence
of nucleic acid taken from a bacteriophage or other particle or
molecular complex displaying a said selected binding member.
[0241] An antibody VH variable domain with the amino acid sequence
of an antibody VH variable domain of a said selected binding member
may be provided in isolated form, as may a binding member
comprising such a VH domain.
[0242] Ability to bind IL-6 may be further tested, also ability to
compete with e.g. a parent antibody molecule or an antibody
molecule 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 or 23
(e.g. in scFv format and/or IgG format, e.g. IgG1) for binding to
IL-6. Ability to neutralize IL-6 may be tested, as discussed
further elsewhere herein.
[0243] A binding member according to the present invention may bind
IL-6 with the affinity of a parent or other antibody molecule, e.g.
scFv, or one of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18,
19, 21, 22 and 23, e.g. IgG1, or with an affinity that is
better.
[0244] A binding member according to the present invention may
neutralise a biological activity of IL-6 with the potency of a
parent or other antibody molecule, one of antibodies 2, 3, 4, 5, 7,
8, 10, 14, 16, 17, 18, 19, 21, 22 and 23 e.g. scFv, or IgGl, or
with a potency that is better.
[0245] Binding affinity and neutralization potency of different
binding members can be compared under appropriate conditions.
[0246] Variants of the VH and VL domains and CDRs of the present
invention, including those for which amino acid sequences are set
out herein, and which can be employed in binding members of the
invention can be obtained by means of methods of sequence
alteration or mutation and screening for antigen binding members
with desired characteristics. Examples of desired characteristics
include but are not limited to: [0247] Increased binding affinity
for antigen relative to known antibodies which are specific for the
antigen [0248] Increased neutralization of an antigen activity
relative to known antibodies which are specific for the antigen if
the activity is known [0249] Specified competitive ability with a
known antibody or ligand to the antigen at a specific molar ratio
[0250] Ability to immunoprecipitate complex [0251] Ability to bind
to a specified epitope [0252] Linear epitope, e.g. peptide sequence
identified using peptide-binding scan as described herein, e.g.
using peptides screened in linear and/or constrained conformation
[0253] Conformational epitope, formed by non-continuous residues
[0254] Ability to modulate a new biological activity of IL-6, or
downstream molecule. Such methods are also provided herein.
[0255] Variants of antibody molecules disclosed herein may be
produced and used in the present invention. Following the lead of
computational chemistry in applying multivariate data analysis
techniques to the structure/property-activity relationships [81]
quantitative activity-property relationships of antibodies can be
derived using well-known mathematical techniques, such as
statistical regression, pattern recognition and classification [82,
83, 84, 85, 86, 87]. The properties of antibodies can be derived
from empirical and theoretical models (for example, analysis of
likely contact residues or calculated physicochemical property) of
antibody sequence, functional and three-dimensional structures and
these properties can be considered singly and in combination.
[0256] An antibody antigen-binding site composed of a VH domain and
a VL domain is typically formed by six loops of polypeptide: three
from the light chain variable domain (VL) and three from the heavy
chain variable domain (VH). Analysis of antibodies of known atomic
structure has elucidated relationships between the sequence and
three-dimensional structure of antibody combining sites [88, 89].
These relationships imply that, except for the third region (loop)
in VH domains, binding site loops have one of a small number of
main-chain conformations: canonical structures. The canonical
structure formed in a particular loop has been shown to be
determined by its size and the presence of certain residues at key
sites in both the loop and in framework regions [88, 89].
[0257] This study of sequence-structure relationship can be used
for prediction of those residues in an antibody of known sequence,
but of an unknown three-dimensional structure, which are important
in maintaining the three-dimensional structure of its CDR loops and
hence maintain binding specificity. These predictions can be backed
up by comparison of the predictions to the output from lead
optimization experiments. In a structural approach, a model can be
created of the antibody molecule [90] using any freely available or
commercial package, such as WAM [91]. A protein visualisation and
analysis software package, such as Insight II (Accelrys, Inc.) or
Deep View [92] may then be used to evaluate possible substitutions
at each position in the CDR. This information may then be used to
make substitutions likely to have a minimal or beneficial effect on
activity.
[0258] The techniques required to make substitutions within amino
acid sequences of CDRs, antibody VH or VL domains and binding
members generally are available in the art. Variant sequences may
be made, with substitutions that may or may not be predicted to
have a minimal or beneficial effect on activity, and tested for
ability to bind and/or neutralize IL-6 and/or for any other desired
property.
[0259] Variable domain amino acid sequence variants of any of the
VH and VL domains whose sequences are specifically disclosed herein
may be employed in accordance with the present invention, as
discussed.
[0260] Variants of VL domains of the invention, and binding members
or antibody molecules comprising them, include VL domains in which
Arginine is not present at Kabat residue 108, e.g. where Kabat
residue 108 is a different residue or is deleted. For example, an
antibody molecule, such as an antibody molecule lacking a constant
domain, e.g. an scFv, may comprise a VL domain having a VL domain
sequence or variant thereof as described herein, in which Arginine
at Kabat residue 108 an amino acid residue other than Arginine or
is deleted.
[0261] A further aspect of the invention is an antibody molecule
comprising a VH domain that has at least 60, 70, 80, 85, 90, 95, 98
or 99% amino acid sequence identity with a VH domain of any of
antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23
shown in the appended sequence listing, and/or comprising a VL
domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino
acid sequence identity with a VL domain of any of antibodies 2, 3,
4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23 shown in the
appended sequence listing. Algorithms that can be used to calculate
% identity of two amino acid sequences include e.g. BLAST [93],
FASTA [94], or the Smith-Waterman algorithm [95], e.g. employing
default parameters.
[0262] Particular variants may include one or more amino acid
sequence alterations (addition, deletion, substitution and/or
insertion of an amino acid residue).
[0263] Alterations may be made in one or more framework regions
and/or one or more CDRs. The alterations normally do not result in
loss of function, so a binding member comprising a thus-altered
amino acid sequence may retain an ability to bind and/or neutralize
IL-6. It may retain the same quantitative binding and/or
neutralizing ability as a binding member in which the alteration is
not made, e.g. as measured in an assay described herein. The
binding member comprising a thus-altered amino acid sequence may
have an improved ability to bind and/or neutralize IL-6. Alteration
may comprise replacing one or more amino acid residue with a
non-naturally occurring or non-standard amino acid, modifying one
or more amino acid residue into a non-naturally occurring or
non-standard form, or inserting one or more non-naturally occurring
or non-standard amino acid into the sequence. Examples of numbers
and locations of alterations in sequences of the invention are
described elsewhere herein. Naturally occurring amino acids include
the "standard" L-amino acids identified as G, A, V, L, I, M, P, F,
W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter
codes. Non-standard amino acids include any other residue that may
be incorporated into a polypeptide backbone or result from
modification of an existing amino acid residue. Non-standard amino
acids may be naturally occurring or non-naturally occurring.
Several naturally occurring non-standard amino acids are known in
the art, such as 4-hydroxyproline, 5-hydroxylysine,
3-methylhistidine, N-acetylserine, etc. [96]. Those amino acid
residues that are derivatised at their N-alpha position will only
be located at the N-terminus of an amino-acid sequence. Normally in
the present invention an amino acid is an L-amino acid, but it may
be a D-amino acid. Alteration may therefore comprise modifying an
L-amino acid into, or replacing it with, a D-amino acid.
Methylated, acetylated and/or phosphorylated forms of amino acids
are also known, and amino acids in the present invention may be
subject to such modification.
[0264] Amino acid sequences in antibody domains and binding members
of the invention may comprise non-natural or non-standard amino
acids described above. Non-standard amino acids (e.g. D-amino
acids) may be incorporated into an amino acid sequence during
synthesis, or by modification or replacement of the "original"
standard amino acids after synthesis of the amino acid
sequence.
[0265] Use of non-standard and/or non-naturally occurring amino
acids increases structural and functional diversity, and can thus
increase the potential for achieving desired IL-6-binding and
neutralizing properties in a binding member of the invention.
Additionally, D-amino acids and analogues have been shown to have
different pharmacokinetic profiles compared with standard L-amino
acids, owing to in vivo degradation of polypeptides having L-amino
acids after administration to an animal e.g. a human, meaning that
D-amino acids are advantageous for some in vivo applications.
[0266] Novel VH or VL regions carrying CDR-derived sequences of the
invention may be generated using random mutagenesis of one or more
selected VH and/or VL genes to generate mutations within the entire
variable domain. Such a technique is described by Gram et al. [97],
who used error-prone PCR. In some embodiments one or two amino acid
substitutions are made within an entire variable domain or set of
CDRs.
[0267] Another method that may be used is to direct mutagenesis to
CDR regions of VH or VL genes. Such techniques are disclosed by
Barbas et al. [98] and Schier et al. [99]. All the above-described
techniques are known as such in the art and the skilled person will
be able to use such techniques to provide binding members of the
invention using routine methodology in the art.
[0268] A further aspect of the invention provides a method for
obtaining an antibody antigen-binding site for IL-6, the method
comprising providing by way of addition, deletion, substitution or
insertion of one or more amino acids in the amino acid sequence of
a VH domain set out herein a VH domain which is an amino acid
sequence variant of the VH domain, optionally combining the VH
domain thus provided with one or more VL domains, and testing the
VH domain or VH/VL combination or combinations to identify a
binding member or an antibody antigen-binding site for IL-6 and
optionally with one or more desired properties, e.g. ability to
neutralize IL-6 activity. Said VL domain may have an amino acid
sequence which is substantially as set out herein. An analogous
method may be employed in which one or more sequence variants of a
VL domain disclosed herein are combined with one or more VH
domains.
[0269] As noted above, a CDR amino acid sequence substantially as
set out herein may be carried as a CDR in a human antibody variable
domain or a substantial portion thereof. The HCDR3 sequences
substantially as set out herein represent embodiments of the
present invention and each of these may be carried as a HCDR3 in a
human heavy chain variable domain or a substantial portion
thereof.
[0270] Variable domains employed in the invention may be obtained
or derived from any germline or rearranged human variable domain,
or may be a synthetic variable domain based on consensus or actual
sequences of known human variable domains. A variable domain can be
derived from a non-human antibody. A CDR sequence of the invention
(e.g. CDR3) may be introduced into a repertoire of variable domains
lacking a CDR (e.g. CDR3), using recombinant DNA technology. For
example, Marks et al. [100] describe methods of producing
repertoires of antibody variable domains in which consensus primers
directed at or adjacent to the 5' end of the variable domain area
are used in conjunction with consensus primers to the third
framework region of human VH genes to provide a repertoire of VH
variable domains lacking a CDR3. Marks et al. further describe how
this repertoire may be combined with a CDR3 of a particular
antibody. Using analogous techniques, the CDR3-derived sequences of
the present invention may be shuffled with repertoires of VH or VL
domains lacking a CDR3, and the shuffled complete VH or VL domains
combined with a cognate VL or VH domain to provide binding members
of the invention. The repertoire may then be displayed in a
suitable host system, such as the phage display system of
WO92/01047, which is herein incorporated by reference in its
entirety, or any of a subsequent large body of literature,
including Kay, Winter & McCafferty [101], so that suitable
binding members may be selected. A repertoire may consist of from
anything from 10.sup.4 individual members upwards, for example at
least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least
10.sup.8, at least 10.sup.9 or at least 10.sup.10 members or more.
Other suitable host systems include, but are not limited to yeast
display, bacterial display, T7 display, viral display, cell
display, ribosome display and covalent display.
[0271] A method of preparing a binding member for IL-6 antigen is
provided, which method comprises: [0272] (a) providing a starting
repertoire of nucleic acids encoding a VH domain which either
include a CDR3 to be replaced or lack a CDR3 encoding region;
[0273] (b) combining said repertoire with a donor nucleic acid
encoding an amino acid sequence substantially as set out herein for
a VH CDR3 such that said donor nucleic acid is inserted into the
CDR3 region in the repertoire, so as to provide a product
repertoire of nucleic acids encoding a VH domain; [0274] (c)
expressing the nucleic acids of said product repertoire; [0275] (d)
selecting a binding member for IL-6; and [0276] (e) recovering said
binding member or nucleic acid encoding it.
[0277] Again, an analogous method may be employed in which a VL
CDR3 of the invention is combined with a repertoire of nucleic
acids encoding a VL domain that either include a CDR3 to be
replaced or lack a CDR3 encoding region.
[0278] Similarly, one or more, or all three CDRs may be grafted
into a repertoire of VH or VL domains that are then screened for a
binding member or binding members for IL-6.
[0279] For example, one or more of the parent or antibody 2, 3, 4,
5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 or 23 HCDR1, HCDR2 and
HCDR3 or the parent or antibody 2, 3, 4, 5, 7, 8, 10, 14, 16, 17,
18, 19, 21, 22 or 23 set of HCDRs may be employed, and/or one or
more of the parent or antibody 2, 3, 4, 5, 7, 8, 10, 14, 16, 17,
18, 19, 21, 22 or 23 LCDR1, LCDR2 and LCDR3 or the parent or
antibody 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 or 23 set
of LCDRs may be employed.
[0280] Similarly, other VH and VL domains, sets of CDRs and sets of
HCDRs and/or sets of LCDRs disclosed herein may be employed.
[0281] A substantial portion of an immunoglobulin variable domain
may comprise at least the three CDR regions, together with their
intervening framework regions. The portion may also include at
least about 50% of either or both of the first and fourth framework
regions, the 50% being the C-terminal 50% of the first framework
region and the N-terminal 50% of the fourth framework region.
Additional residues at the N-terminal or C-terminal end of the
substantial part of the variable domain may be those not normally
associated with naturally occurring variable domain regions. For
example, construction of binding members of the present invention
made by recombinant DNA techniques may result in the introduction
of N- or C-terminal residues encoded by linkers introduced to
facilitate cloning or other manipulation steps. Other manipulation
steps include the introduction of linkers to join variable domains
of the invention to further protein sequences including antibody
constant regions, other variable domains (for example in the
production of diabodies) or detectable/functional labels as
discussed in more detail elsewhere herein.
[0282] Although in some aspects of the invention, binding members
comprise a pair of VH and VL domains, single binding domains based
on either VH or VL domain sequences form further aspects of the
invention. It is known that single immunoglobulin domains,
especially VH domains, are capable of binding target antigens in a
specific manner. For example, see the discussion of dAbs above. In
the case of either of the single binding domains, these domains may
be used to screen for complementary domains capable of forming a
two-domain binding member able to bind IL-6. This may be achieved
by phage display screening methods using the so-called hierarchical
dual combinatorial approach as disclosed in WO92/01047, herein
incorporated by reference in its entirety, in which an individual
colony containing either an H or L chain clone is used to infect a
complete library of clones encoding the other chain (L or H) and
the resulting two-chain binding member is selected in accordance
with phage display techniques, such as those described in that
reference. This technique is also disclosed in Marks et al, ibid.
[100].
[0283] Binding members of the present invention may further
comprise antibody constant regions or parts thereof, e.g. human
antibody constant regions or parts thereof. For example, a VL
domain may be attached at its C-terminal end to antibody light
chain constant domains including human C.kappa. or C.lamda.,
chains. Similarly, a binding member based on a VH domain may be
attached at its C-terminal end to all or part (e.g. a CH1 domain)
of an immunoglobulin heavy chain derived from any antibody isotype,
e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes,
particularly IgG1 and IgG4. IgG1 is advantageous, due to its
effector function and ease of manufacture. Any synthetic or other
constant region variant that has these properties and stabilizes
variable regions may also be useful in the present invention.
[0284] Binding members of the invention may be labelled with a
detectable or functional label. Thus, a binding member or antibody
molecule can be present in the form of an immunoconjugate so as to
obtain a detectable and/or quantifiable signal. An immunoconjugates
may comprise an antibody molecule of the invention conjugated with
detectable or functional label. A label can be any molecule that
produces or can be induced to produce a signal, including but not
limited to fluorescers, radiolabels, enzymes, chemiluminescers or
photosensitizers. Thus, binding may be detected and/or measured by
detecting fluorescence or luminescence, radioactivity, enzyme
activity or light absorbance.
[0285] Suitable labels include, by way of illustration and not
limitation, [0286] enzymes, such as alkaline phosphatase,
glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase,
glucose oxydase, glucose amylase, carbonic anhydrase,
acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase
e.g. horseradish peroxidase; [0287] dyes; [0288] fluorescent labels
or fluorescers, such as fluorescein and its derivatives,
derivatives, fluorochrome, rhodamine compounds and derivatives,
GFP, (GFP for "Green Flourescent Protein"), dansyl, unbelliferone,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and
fluorescamine; fluorophores such as lanthanide phthaldehyde, and
fluorescamine; fluorophores such as lanthanide cryptates and
chelates e.g. Europium etc (Perkin Elmer and Cis Biointernational),
[0289] chemoluminescent labels or chemiluminescers, such as
isoluminol, luminol and the dioxetanes; [0290] bio-luminescent
labels, such as luciferase and luciferin; [0291] sensitizers;
[0292] coenzymes; [0293] enzyme substrates; [0294] radiolabels
including but not limited to bromine77, carbon14, cobalt57,
fluorine8, gallium67, gallium 68, hydrogen3 (tritium), indium111,
indium 113m, iodine123m, iodine125, iodine126, iodine131,
iodine133, mercury107, mercury203, phosphorous32, rhenium99m,
rhenium101, rhenium105, ruthenium95, ruthenium97, ruthenium103,
ruthenium105, scandium47, selenium75, sulphur35, technetium99,
technetium99m, tellurium121m, tellurium122m, tellurium125m,
thulium165, thulium167, thulium168, yttrium199 and other
radiolabels mentioned herein; [0295] particles, such as latex or
carbon particles; metal sol; crystallite; liposomes; cells, etc.,
which may be further labelled with a dye, catalyst or other
detectable group; [0296] molecules such as biotin, digoxygenin or
5-bromodeoxyuridine; [0297] toxin moieties, such as for example a
toxin moiety selected from a group of Pseudomonas exotoxin (PE or a
cytotoxic fragment or mutant thereof), Diptheria toxin or a
cytotoxic fragment or mutant thereof, a botulinum toxin A, B, C, D,
E or F, ricin or a cytotoxic fragment thereof e.g. ricin A, abrin
or a cytotoxic fragment thereof, saporin or a cytotoxic fragment
thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof
and bryodin 1 or a cytotoxic fragment thereof.
[0298] Suitable enzymes and coenzymes are disclosed in Litman, et
al., U.S. Pat. No. 4,275,149, and Boguslaski, et al., U.S. Pat. No.
4,318,980, each of which are herein incorporated by reference in
their entireties. Suitable fluorescers and chemiluminescers are
disclosed in Litman, et al., U.S. Pat. No. 4,275,149, which is
incorporated herein by reference in its entirety. Labels further
include chemical moieties, such as biotin that may be detected via
binding to a specific cognate detectable moiety, e.g. labelled
avidin or streptavidin. Detectable labels may be attached to
antibodies of the invention using conventional chemistry known in
the art.
[0299] Immunoconjugates or their functional fragments can be
prepared by methods known to the person skilled in the art. They
can be coupled to enzymes or to fluorescent labels directly or by
the intermediary of a spacer group or of a linking group, such as a
polyaldehyde, like glutaraldehyde, ethylenediaminetetraacetic acid
(EDTA), diethylene-triaminepentaacetic acid (DPTA), or in the
presence of coupling agents, such as those mentioned above for the
therapeutic conjugates. Conjugates containing labels of fluorescein
type can be prepared by reaction with an isothiocyanate.
[0300] The methods known to the person skilled in the art existing
for coupling the therapeutic radioisotopes to the antibodies either
directly or via a chelating agent, such as EDTA, DTPA mentioned
above can be used for the radioelements which can be used in
diagnosis. It is likewise possible to perform labelling with
sodium125 by the chloramine T method [102] or else with
technetium99m by the technique of Crockford et al., (U.S. Pat. No.
4,424,200, herein incorporated by reference in its entirety) or
attached via DTPA as described by Hnatowich (U.S. Pat. No.
4,479,930, herein incorporated by reference in its entirety).
[0301] There are numerous methods by which the label can produce a
signal detectable by external means, for example, by visual
examination, electromagnetic radiation, heat, and chemical
reagents. The label can also be bound to another binding member
that binds the antibody of the invention, or to a support.
[0302] The label can directly produce a signal, and therefore,
additional components are not required to produce a signal.
Numerous organic molecules, for example fluorescers, are able to
absorb ultraviolet and visible light, where the light absorption
transfers energy to these molecules and elevates them to an excited
energy state. This absorbed energy is then dissipated by emission
of light at a second wavelength. This second wavelength emission
may also transfer energy to a labelled acceptor molecule, and the
resultant energy dissipated from the acceptor molecule by emission
of light for example fluorescence resonance energy transfer (FRET).
Other labels that directly produce a signal include radioactive
isotopes and dyes.
[0303] Alternately, the label may need other components to produce
a signal, and the signal producing system would then include all
the components required to produce a measurable signal, which may
include substrates, coenzymes, enhancers, additional enzymes,
substances that react with enzymic products, catalysts, activators,
cofactors, inhibitors, scavengers, metal ions, and a specific
binding substance required for binding of signal generating
substances. A detailed discussion of suitable signal producing
systems can be found in Ullman, et al. U.S. Pat. No. 5,185,243,
which is herein incorporated herein by reference in its entirety.
The present invention provides a method comprising causing or
allowing binding of a binding member as provided herein to IL-6. As
noted, such binding may take place in vivo, e.g. following
administration of a binding member, or nucleic acid encoding a
binding member, or it may take place in vitro, for example in
ELISA, Western blotting, immunocytochemistry, immunoprecipitation,
affinity chromatography, and biochemical or cell-based assays, such
as a TF-1 cell proliferation assay.
[0304] The present invention also provides for measuring levels of
antigen directly, by employing a binding member according to the
invention for example in a biosensor system. For instance, the
present invention comprises a method of detecting and/or measuring
binding to IL-6, comprising, (i) exposing said binding member to
IL-6 and (ii) detecting binding of said binding member to IL-6,
wherein binding is detected using any method or detectable label
described herein. This, and any other binding detection method
described herein, may be interpreted directly by the person
performing the method, for instance, by visually observing a
detectable label. Alternatively, this method, or any other binding
detection method described herein, may produce a report in the form
of an autoradiograph, a photograph, a computer printout, a flow
cytometry report, a graph, a chart, a test tube or container or
well containing the result, or any other visual or physical
representation of a result of the method.
[0305] The amount of binding of binding member to IL-6 may be
determined. Quantitation may be related to the amount of the
antigen in a test sample, which may be of diagnostic interest.
Screening for IL-6 binding and/or the quantitation thereof may be
useful, for instance, in screening patients for diseases or
disorders referred to herein and/or any other disease or disorder
involving aberrant IL-6 expression and/or activity.
[0306] A diagnostic method of the invention may comprise (i)
obtaining a tissue or fluid sample from a subject, (ii) exposing
said tissue or fluid sample to one or more binding members of the
present invention; and (iii) detecting bound IL-6 as compared with
a control sample, wherein an increase in the amount of IL-6 binding
as compared with the control may indicate an aberrant level of IL-6
expression or activity. Tissue or fluid samples to be tested
include blood, serum, urine, biopsy material, tumours, or any
tissue suspected of containing aberrant IL-6 levels. Subjects
testing positive for aberrant IL-6 levels or activity may also
benefit from the treatment methods disclosed later herein. Those
skilled in the art are able to choose a suitable mode of
determining binding of the binding member to an antigen according
to their preference and general knowledge, in light of the methods
disclosed herein.
[0307] The reactivities of binding members in a sample may be
determined by any appropriate means. Radioimmunoassay (MA) is one
possibility. Radioactive labelled antigen is mixed with unlabeled
antigen (the test sample) and allowed to bind to the binding
member. Bound antigen is physically separated from unbound antigen
and the amount of radioactive antigen bound to the binding member
determined. The more antigen there is in the test sample the less
radioactive antigen will bind to the binding member. A competitive
binding assay may also be used with non radioactive antigen, using
antigen or an analogue linked to a reporter molecule. The reporter
molecule may be a fluorochrome, phosphor or laser dye with
spectrally isolated absorption or emission characteristics.
Suitable fluorochromes include fluorescein, rhodamine,
phycoerythrin and Texas Red, and lanthanide chelates or cryptates.
Suitable chromogenic dyes include diaminobenzidine.
[0308] Other reporters include macromolecular colloidal particles
or particulate material, such as latex beads that are colored,
magnetic or paramagnetic, and biologically or chemically active
agents that can directly or indirectly cause detectable signals to
be visually observed, electronically detected or otherwise
recorded. These molecules may be enzymes, which catalyze reactions
that develop, or change colours or cause changes in electrical
properties, for example. They may be molecularly excitable, such
that electronic transitions between energy states result in
characteristic spectral absorptions or emissions. They may include
chemical entities used in conjunction with biosensors.
Biotin/avidin or biotin/streptavidin and alkaline phosphatase
detection systems may be employed.
[0309] The signals generated by individual binding member-reporter
conjugates may be used to derive quantifiable absolute or relative
data of the relevant binding member binding in samples (normal and
test).
[0310] A kit comprising a binding member according to any aspect or
embodiment of the present invention is also provided as an aspect
of the present invention. In the kit, the binding member may be
labelled to allow its reactivity in a sample to be determined, e.g.
as described further below. Further the binding member may or may
not be attached to a solid support. Components of a kit are
generally sterile and in sealed vials or other containers. Kits may
be employed in diagnostic analysis or other methods for which
binding members are useful. A kit may contain instructions for use
of the components in a method, e.g. a method in accordance with the
present invention. Ancillary materials to assist in or to enable
performing such a method may be included within a kit of the
invention. The ancillary materials include a second, different
binding member which binds to the first binding member and is
conjugated to a detectable label (e.g., a fluorescent label,
radioactive isotope or enzyme). Antibody-based kits may also
comprise beads for conducting an immunoprecipitation. Each
component of the kits is generally in its own suitable container.
Thus, these kits generally comprise distinct containers suitable
for each binding member. Further, the kits may comprise
instructions for performing the assay and methods for interpreting
and analyzing the data resulting from the performance of the
assay.
[0311] The present invention also provides the use of a binding
member as above for measuring antigen levels in a competition
assay, that is to say a method of measuring the level of antigen in
a sample by employing a binding member as provided by the present
invention in a competition assay. This may be where the physical
separation of bound from unbound antigen is not required. Linking a
reporter molecule to the binding member so that a physical or
optical change occurs on binding is one possibility. The reporter
molecule may directly or indirectly generate detectable signals,
which may be quantifiable. The linkage of reporter molecules may be
directly or indirectly, covalently, e.g. via a peptide bond or
non-covalently. Linkage via a peptide bond may be as a result of
recombinant expression of a gene fusion encoding antibody and
reporter molecule.
[0312] In various aspects and embodiments, the present invention
extends to a binding member that competes for binding to IL-6 with
any binding member defined herein, e.g. the parent antibody or any
of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and
23, e.g. in IgG1 format. Competition between binding members may be
assayed easily in vitro, for example by tagging a specific reporter
molecule to one binding member which can be detected in the
presence of other untagged binding member(s), to enable
identification of binding members which bind the same epitope or an
overlapping epitope. Competition may be determined for example
using ELISA in which IL-6 is immobilized to a plate and a first
tagged or labelled binding member along with one or more other
untagged or unlabeled binding members is added to the plate.
Presence of an untagged binding member that competes with the
tagged binding member is observed by a decrease in the signal
emitted by the tagged binding member.
[0313] For example, the present invention includes a method of
identifying an IL-6 binding compound, comprising (i) immobilizing
IL-6 to a support, (ii) contacting said immobilized IL-6
simultaneously or in a step-wise manner with at least one tagged or
labelled binding member according to the invention and one or more
untagged or unlabeled test binding compounds, and (iii) identifying
a new IL-6 binding compound by observing a decrease in the amount
of bound tag from the tagged binding member. Such methods can be
performed in a high-throughput manner using a multiwell or array
format. Such assays may be also be performed in solution. See, for
instance, U.S. Pat. No. 5,814,468, which is herein incorporated by
reference in its entirety. As described above, detection of binding
may be interpreted directly by the person performing the method,
for instance, by visually observing a detectable label, or a
decrease in the presence thereof. Alternatively, the binding
methods of the invention may produce a report in the form of an
autoradiograph, a photograph, a computer printout, a flow cytometry
report, a graph, a chart, a test tube or container or well
containing the result, or any other visual or physical
representation of a result of the method.
[0314] Competition assays can also be used in epitope mapping. In
one instance epitope mapping may be used to identify the epitope
bound by an IL-6 binding member which optionally may have optimized
neutralizing and/or modulating characteristics. Such an epitope can
be linear or conformational. A conformational epitope can comprise
at least two different fragments of IL-6, wherein said fragments
are positioned in proximity to each other when IL-6 is folded in
its tertiary or quaternary structure to form a conformational
epitope which is recognized by an inhibitor of IL-6, such as an
IL-6-binding member. In testing for competition a peptide fragment
of the antigen may be employed, especially a peptide including or
consisting essentially of an epitope of interest. A peptide having
the epitope sequence plus one or more amino acids at either end may
be used. Binding members according to the present invention may be
such that their binding for antigen is inhibited by a peptide with
or including the sequence given.
[0315] The present invention further provides an isolated nucleic
acid encoding a binding member of the present invention. Nucleic
acid may include DNA and/or RNA. In one, the present invention
provides a nucleic acid that codes for a CDR or set of CDRs or VH
domain or VL domain or antibody antigen-binding site or antibody
molecule, e.g. scFv or IgG1, of the invention as defined above.
[0316] The present invention also provides constructs in the form
of plasmids, vectors, transcription or expression cassettes which
comprise at least one polynucleotide as above.
[0317] The present invention also provides a recombinant host cell
that comprises one or more constructs as above. A nucleic acid
encoding any CDR or set of CDRs or VH domain or VL domain or
antibody antigen-binding site or antibody molecule, e.g. scFv or
IgG1 as provided, itself forms an aspect of the present invention,
as does a method of production of the encoded product, which method
comprises expression from encoding nucleic acid therefor.
Expression may conveniently be achieved by culturing under
appropriate conditions recombinant host cells containing the
nucleic acid. Following production by expression a VH or VL domain,
or binding member may be isolated and/or purified using any
suitable technique, then used as appropriate.
[0318] Nucleic acid according to the present invention may comprise
DNA or RNA and may be wholly or partially synthetic. Reference to a
nucleotide sequence as set out herein encompasses a DNA molecule
with the specified sequence, and encompasses a RNA molecule with
the specified sequence in which U is substituted for T, unless
context requires otherwise.
[0319] A yet further aspect provides a method of production of an
antibody VH variable domain, the method including causing
expression from encoding nucleic acid. Such a method may comprise
culturing host cells under conditions for production of said
antibody VH variable domain.
[0320] Analogous methods for production of VL variable domains and
binding members comprising a VH and/or VL domain are provided as
further aspects of the present invention.
[0321] A method of production may comprise a step of isolation
and/or purification of the product. A method of production may
comprise formulating the product into a composition including at
least one additional component, such as a pharmaceutically
acceptable excipient.
[0322] Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host cells
include bacteria, mammalian cells, plant cells, filamentous fungi,
yeast and baculovirus systems and transgenic plants and
animals.
[0323] The expression of antibodies and antibody fragments in
prokaryotic cells is well established in the art. For a review, see
for example Pluckthun [103]. A common bacterial host is E.
coli.
[0324] Expression in eukaryotic cells in culture is also available
to those skilled in the art as an option for production of a
binding member [104, 105, 106]. Mammalian cell lines available in
the art for expression of a heterologous polypeptide include
Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney
cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human
embryonic kidney cells, human embryonic retina cells and many
others.
[0325] Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator sequences, polyadenylation sequences, enhancer
sequences, marker genes and other sequences as appropriate. Vectors
may be plasmids e.g. phagemid, or viral e.g. `phage, as appropriate
[107]. Many known techniques and protocols for manipulation of
nucleic acid, for example in preparation of nucleic acid
constructs, mutagenesis, sequencing, introduction of DNA into cells
and gene expression, and analysis of proteins, are described in
detail in Ausubel et al. [108].
[0326] A further aspect of the present invention provides a host
cell containing nucleic acid as disclosed herein. Such a host cell
may be in vitro and may be in culture. Such a host cell may be in
vivo. In vivo presence of the host cell may allow intra-cellular
expression of the binding members of the present invention as
"intrabodies" or intra-cellular antibodies. Intrabodies may be used
for gene therapy.
[0327] A still further aspect provides a method comprising
introducing nucleic acid of the invention into a host cell. The
introduction may employ any available technique. For eukaryotic
cells, suitable techniques may include calcium phosphate
transfection, DEAE-Dextran, electroporation, liposome-mediated
transfection and transduction using retrovirus or other virus, e.g.
vaccinia or, for insect cells, baculovirus. Introducing nucleic
acid in the host cell, in particular a eukaryotic cell may use a
viral or a plasmid based system. The plasmid system may be
maintained episomally or may be incorporated into the host cell or
into an artificial chromosome. Incorporation may be either by
random or targeted integration of one or more copies at single or
multiple loci. For bacterial cells, suitable techniques may include
calcium chloride transformation, electroporation and transfection
using bacteriophage.
[0328] The introduction may be followed by causing or allowing
expression from the nucleic acid, e.g. by culturing host cells
under conditions for expression of the gene. The purification of
the expressed product may be achieved by methods known to one of
skill in the art.
[0329] Nucleic acid of the invention may be integrated into the
genome (e.g. chromosome) of the host cell. Integration may be
promoted by inclusion of sequences that promote recombination with
the genome, in accordance with standard techniques.
[0330] The present invention also provides a method that comprises
using a construct as stated above in an expression system in order
to express a binding member or polypeptide as above.
[0331] There is evidence for involvement of IL-6 in a variety of
disorders, as discussed elsewhere herein. The binding members of
the present invention may therefore be used in a method of
diagnosis or treatment of a disorder associated with IL-6. Such a
disorder may for example be an inflammatory and/or autoimmune
disorder such as for example, rheumatoid arthritis, osteoarthritis,
cachexia, chronic obstructive pulmonary disease, Juvenile
idiopathic arthritis, asthma, systemic lupus erythematosus,
inflammatory bowel disease, Crohn's disease or atherosclerosis. A
binding member of the present invention may also be used to treat a
disorder such as a tumour and/or cancer.
[0332] Binding members of the present invention may also be used in
method of diagnosis or treatment of at least one IL-6 related
disease, in a patient, animal, organ, tissue or cell, including,
but not limited to:--
[0333] (the respiratory tract) obstructive airways diseases
including chronic obstructive pulmonary disease (COPD); asthma,
such as bronchial, allergic, intrinsic, extrinsic and dust asthma,
particularly chronic or inveterate asthma (e.g. late asthma and
airways hyper-responsiveness); bronchitis; acute-, allergic-,
atrophic rhinitis and chronic rhinitis including rhinitis caseosa,
hypertrophic rhinitis, rhinitis purulenta, rhinitis sicca and
rhinitis medicamentosa; membranous rhinitis including croupous,
fibrinous and pseudomembranous rhinitis and scrofoulous rhinitis;
seasonal rhinitis including rhinitis nervosa (hay fever) and
vasomotor rhinitis, sinusitis, idiopathic pulmonary fibrosis (IPF);
sarcoidosis, farmer's lung and related diseases, adult respiratory
distress syndrome, hypersensitivity pneumonitis, fibroid lung and
idiopathic interstitial pneumonia;
[0334] (bone and joints) rheumatoid arthritis, juvenile chronic
arthritis, systemic onset juvenile arthritis, seronegative
spondyloarthropathies (including ankylosing spondylitis, psoriatic
arthritis and Reiter's disease), Behcet's disease, Siogren's
syndrome and systemic sclerosis, gout, osteoporosis and
osteoarthritis;
[0335] (skin) psoriasis, atopical dermatitis, contact dermatitis
and other eczmatous dermatoses, allergic contact dermatitis,
seborrhoetic dermatitis, Lichen planus, scleroderma, Pemphigus,
bullous pemphigoid, Epidermolysis bullosa, urticaria, angiodermas,
vasculitides, erythemas, cutaneous eosinophilias, uveitis, Alopecia
areata, allergic conjunctivitis and vernalvemal conjunctivitis;
[0336] (gastrointestinal tract) gastric ulcer, Coeliac disease,
proctitis, eosinopilic gastro-enteritis, mastocytosis, inflammatory
bowel disease, Crohn's disease, ulcerative colitis,
antiphospholipid syndrome)), food-related allergies which have
effects remote from the gut, e.g., migraine, rhinitis and
eczema;
[0337] (other tissues and systemic disease) cachexia, multiple
sclerosis, atherosclerosis, Acquired Immunodeficiency Syndrome
(AIDS), mesangial proliferative glomerulonephritis, nephrotic
syndrome, nephritis, glomerular nephritis, acute renal failure,
hemodialysis, uremia, localised or discoid lupus erythematosus,
systemic lupus erythematosus, Castleman's Disease, Hashimoto's
thyroiditis, myasthenia gravis, type I diabetes, type B
insulin-resistant diabetes, sickle cell anaemia,
iridocyclitis/uveitis/optic neuritis, nephritic syndrome,
eosinophilia fascitis, hyper IgE syndrome, systemic
vasculitis/wegener's granulomatosis, orchitis/vasectomy reversal
procedures, lepromatous leprosy, alcohol-induced hepatitis, sezary
syndrome and idiopathic thrombocytopenia purpura; post-operative
adhesions, nephrosis, systemic inflammatory response syndrome,
sepsis syndrome, gram positive sepsis, gram negative sepsis,
culture negative sepsis, fungal sepsis, neutropenic fever, acute
pancreatitis, urosepsis, Graves disease, Raynaud's disease,
antibody-mediatated cytotoxicity, type III hypersensitivity
reactions, POEMS syndrome (polyneuropathy, organomegaly,
endocrinopathy, monoclonal gammopathy, and skin changes syndrome),
mixed connective tissue disease, idiopathic Addison's disease,
diabetes mellitus, chronic active hepatitis, primary billiary
cirrhosis, vitiligo, post-MI (cardiotomy) syndrome, type IV
hypersensitivity, granulomas due to intracellular organisms,
Wilson's disease, hemachromatosis, alpha-I-antitrypsin deficiency,
diabetic retinopathy, hashimoto's thyroiditis,
hypothalamic-pituitary-adrenal axis evaluation, thyroiditis,
encephalomyelitis, neonatal chronic lung disease, familial
hematophagocytic lymphohistiocytosis, alopecia, radiation therapy
(e.g., including but not limited to asthenia, anemia, cachexia, and
the like), chronic salicylate intoxication, sleep apnea, obesity,
heart failure, and meningococcemia;
[0338] (allograft rejection) acute and chronic following, for
example, transplantation of kidney, heart, liver, lung, pancreas,
bone marrow, bone, small bowel, skin, cartilage and cornea; and
chronic graft versus host disease;
[0339] (malignant disease) leukaemia, acute lymphoblastic leukaemia
(ALL), acute leukaemia, T-cell, B-cell, or FAB ALL, chromic
myelocytic leukaemia (CIVIL), acute myeloid leukaemia (AML),
chronic lymphocytic leukaemia (CLL), hairy cell leukaemia,
myelodyplastic syndrome (MDS), any lymphoma, Hodgkin's disease,
non-hodgkin's lymphoma, any malignant lymphoma, Burkitt's lymphoma,
multiple myeloma, Kaposi's sarcoma, renal cell carcinoma,
colorectal carcinoma, prostatic carcinoma, pancreatic carcinoma,
nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic
syndrome/hypercalcemia of malignancy, solid tumors,
adenocarcinomas, sarcomas, malignant melanoma, hemangioma,
metastatic disease, cancer related bone resorption, cancer related
bone pain; the suppression of cancer metastasis; the amelioration
of cancer cachexia;
[0340] Cystic fibrosis, stroke, re-perfusion injury in the heart,
brain, peripheral limbs and other organs;
[0341] Burn wounds, trauma/haemorrhage, ionizing radiation
exposure, chronic skin ulcers;
[0342] Reproductive Diseases (e.g. Disorders of ovulation,
menstruation and implantation, pre-term labour, pre-eclampsia,
endometriosis); (Infections) acute or chronic bacterial infection,
acute and chronic parasitic or infectious processes, including
bacterial, viral and fungal infections, HIV infection/HIV
neuropathy, meningitis, hepatitis (A, B or C, or other viral
hepatitis the like), septic arthritis, peritonitis, pneumonia,
epiglottitis, E. coli 0157:h7, hemolytic uremic syndrome/thrombotic
thrombocytopenic purpura, malaria, dengue hemorrhagic fever,
leishmaniasis, leprosy, toxic shock syndrome, streptococcal
myositis, gas gangrene, Mycobacterium tuberculosis, Mycobacterium
avium intracellulare, Pneumocystis carinii pneumonia, pelvic
inflammatory disease, orchitis/epidydimitis, legionella, Lyme
disease, influenza a, epstein-barr virus, vital-associated
hemaphagocytic syndrome, viral encephalitis/aseptic meningitis, and
the like.
[0343] Accordingly, the invention provides a method of treating an
IL-6 related disorder, comprising administering to a patient in
need thereof an effective amount of one or more binding members of
the present invention alone or in a combined therapeutic regimen
with another appropriate medicament known in the art or described
herein.
[0344] Evidence for involvement of IL-6 in certain disorders is
summarised elsewhere herein. In addition, the data presented herein
further indicates that binding members of the invention can be used
to treat such disorders, including preventative treatment and
reduction of severity of the disorders. Accordingly, the invention
provides a method of treating or reducing the severity of at least
one symptom of any of the disorders mentioned herein, comprising
administering to a patient in need thereof an effective amount of
one or more binding members of the present invention alone or in a
combined therapeutic regimen with another appropriate medicament
known in the art or described herein such that the severity of at
least one symptom of any of the above disorders is reduced.
[0345] Thus, the binding members of the present invention are
useful as therapeutic agents in the treatment of diseases or
disorders involving IL-6 and/or IL-6Ra expression and/or activity,
especially aberrant expression/activity. A method of treatment may
comprise administering an effective amount of a binding member of
the invention to a patient in need thereof, wherein aberrant
expression and/or activity of IL-6 and/or IL-6Ra is decreased. A
method of treatment may comprise (i) identifying a patient
demonstrating aberrant IL-6:IL-6Ra levels or activity, for instance
using the diagnostic methods described above, and (ii)
administering an effective amount of a binding member of the
invention to the patient, wherein aberrant expression and/or
activity of IL-6Ra and/or IL-6 is decreased. An effective amount
according to the invention is an amount that decreases the aberrant
expression and/or activity of IL-6 and/or IL-6Ra so as to decrease
or lessen the severity of at least one symptom of the particular
disease or disorder being treated, but not necessarily cure the
disease or disorder.
[0346] The invention also provides a method of antagonising at
least one effect of IL-6, comprising contacting with or
administering an effective amount of one or more binding members of
the present invention such that said at least one effect of IL-6 is
antagonised. Effects of IL-6 that may be antagonised by the methods
of the invention include IL-6 binding to gp130, and downstream
effects that arise as a consequence of this binding. Accordingly,
further aspects of the invention provide methods of treatment
comprising administration of a binding member as provided,
pharmaceutical compositions comprising such a binding member, and
use of such a binding member in the manufacture of a medicament for
administration, for example in a method of making a medicament or
pharmaceutical composition comprising formulating the binding
member with a pharmaceutically acceptable excipient. A
pharmaceutically acceptable excipient may be a compound or a
combination of compounds entering into a pharmaceutical composition
not provoking secondary reactions and which allows, for example,
facilitation of the administration of the active compound(s), an
increase in its lifespan and/or in its efficacy in the body, an
increase in its solubility in solution or else an improvement in
its conservation. These pharmaceutically acceptable vehicles are
well known and will be adapted by the person skilled in the art as
a function of the nature and of the mode of administration of the
active compound(s) chosen.
[0347] Binding members of the present invention will usually be
administered in the form of a pharmaceutical composition, which may
comprise at least one component in addition to the binding member.
Thus pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may comprise, in addition to active ingredient, a pharmaceutically
acceptable excipient, carrier, buffer, stabilizer or other
materials well known to those skilled in the art. Such materials
should be non-toxic and should not interfere with the efficacy of
the active ingredient. The precise nature of the carrier or other
material will depend on the route of administration, which may be
oral, inhaled, intra-tracheal, topical, intra-vesicular or by
injection, as discussed below.
[0348] Pharmaceutical compositions for oral administration, such as
for example single domain antibody molecules (e.g.
"Nanobodies.TM.") etc. are also envisaged in the present invention.
Such oral formulations may be in tablet, capsule, powder, liquid or
semi-solid form. A tablet may comprise a solid carrier, such as
gelatin or an adjuvant. Liquid pharmaceutical compositions
generally comprise a liquid carrier, such as water, petroleum,
animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols, such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0349] For intra-venous injection, or injection at the site of
affliction, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and
has suitable pH, isotonicity and stability. Those of relevant skill
in the art are well able to prepare suitable solutions using, for
example, isotonic vehicles, such as Sodium Chloride Injection,
Ringer's Injection, Lactated Ringer's Injection. Preservatives,
stabilizers, buffers, antioxidants and/or other additives may be
employed as required including buffers such as phosphate, citrate
and other organic acids; antioxidants, such as ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride;
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens, such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight
polypeptides; proteins, such as serum albumin, gelatin or
immunoglobulins; hydrophilic polymers, such as
polyvinylpyrrolidone; amino acids, such as glycine, glutamine,
asparagines, histidine, arginine, or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose or
dextrins; chelating agents, such as EDTA; sugars, such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions, such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants, such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0350] Binding members of the present invention may be formulated
in liquid, semi-solid or solid forms depending on the
physicochemical properties of the molecule and the route of
delivery. Formulations may include excipients, or combinations of
excipients, for example: sugars, amino acids and surfactants.
Liquid formulations may include a wide range of antibody
concentrations and pH. Solid formulations may be produced by
lyophilisation, spray drying, or drying by supercritical fluid
technology, for example. Formulations of binding members will
depend upon the intended route of delivery: for example,
formulations for pulmonary delivery may consist of particles with
physical properties that ensure penetration into the deep lung upon
inhalation; topical formulations (e.g. for treatment of scarring,
e.g. dermal scarring) may include viscosity modifying agents, which
prolong the time that the drug is resident at the site of action. A
binding member may be prepared with a carrier that will protect the
binding member against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are known to those skilled in the art [109].
[0351] Treatment may be given orally (such as for example single
domain antibody molecules (e.g. "Nanobodies.TM.")) by injection
(for example, subcutaneously, intra-articular, intra-venously,
intra-peritoneal, intra-arterial or intra-muscularly), by
inhalation, intra-tracheal, by the intra-vesicular route
(instillation into the urinary bladder), or topically (for example
intra-ocular, intra-nasal, rectal, into wounds, on skin). The
treatment may be administered by pulse infusion, particularly with
declining doses of the binding member. The route of administration
can be determined by the physicochemical characteristics of the
treatment, by special considerations for the disease or by the
requirement to optimize efficacy or to minimize side-effects. One
particular route of administration is intra-venous. Another route
of administering pharmaceutical compositions of the present
invention is subcutaneously. It is envisaged that treatment will
not be restricted to use in the clinic. Therefore, subcutaneous
injection using a needle-free device is also advantageous.
[0352] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated.
[0353] A binding member of the invention may be used as part of a
combination therapy in conjunction with an additional medicinal
component. Combination treatments may be used to provide
significant synergistic effects, particularly the combination of a
binding member of the invention with one or more other drugs. A
binding member of the invention may be administered concurrently or
sequentially or as a combined preparation with another therapeutic
agent or agents, for the treatment of one or more of the conditions
listed herein.
[0354] A binding member of the invention may be used as a
chemosensitiser whereby it can increase therapeutic efficacy of
cytotoxic agents, and may thus be provided for administration in
combination with one or more cytotoxic agents, either
simultaneously or sequentially. The binding member may also be used
as a radio sensitiser whereby it can improve efficacy of radiation,
and may thus be provided for administration in combination with
radiation, either simultaneously or sequentially.
[0355] A binding member according to the present invention may be
provided in combination or addition with one or more of the
following agents: [0356] a cytokine or agonist or antagonist of
cytokine function (e.g. an agent which acts on cytokine signalling
pathways, such as a modulator of the SOCS system), such as an
alpha-, beta- and/or gamma-interferon; insulin-like growth factor
type I (IGF-1), its receptors and associated binding proteins;
interleukins (IL), e.g. one or more of IL-1 to -33, and/or an
interleukin antagonist or inhibitor, such as anakinra; inhibitors
of receptors of interleukin family members or inhibitors of
specific subunits of such receptors, a tumour necrosis factor alpha
(TNF-a) inhibitor, such as an anti-TNF monoclonal antibodies (for
example infliximab, adalimumab and/or CDP-870) and/or a TNF
receptor antagonist, e.g. an immunoglobulin molecule (such as
etanercept) and/or a low-molecular-weight agent, such as
pentoxyfylline; [0357] a modulator of B cells, e.g. a monoclonal
antibody targeting B-lymphocytes (such as CD20 (rituximab) or
MRA-aIL16R) or 1-lymphocytes (e.g. CTLA4-Ig, HuMax 11-15 or
Abatacept); [0358] a modulator that inhibits osteoclast activity,
for example an antibody to RANKL; [0359] a modulator of chemokine
or chemokine receptor function, such as an antagonist of CCR1,
CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10
and CCR11 (for the C-C family); CXCR1, CXCR2, CXCR3, CXCR4 and
CXCR5 and CXCR6 (for the C-X-C family) and CX.sub.3CR1 for the
C-X.sub.3-C family; [0360] an inhibitor of matrix metalloproteases
(MMPs), i.e. one or more of the stromelysins, the collagenases and
the gelatinases as well as aggrecanase, especially collagenase-1
(MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13),
stromelysin-1 (MMP-3), stromelysin-2 (MMP-10) and/or stromelysin-3
(MMP-11) and/or MMP-9 and/or MMP-12, e.g. an agent such as
doxycycline; [0361] a leukotriene biosynthesis inhibitor,
5-lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activating
protein (FLAP) antagonist, such as zileuton; ABT-761; fenleuton;
tepoxalin; Abbott-79175; Abbott-85761;
N-(5-substituted)-thiophene-2-alkylsulfonamides;
2,6-di-tert-butylphenolhydrazones; methoxytetrahydropyrans such as
Zeneca ZD-2138; the compound SB-210661; a pyridinyl-substituted
2-cyanonaphthalene compound, such as L-739,010; a 2-cyanoquinoline
compound, such as L-746,530; indole and/or a quinoline compound,
such as MK-591, MK-886 and/or BAY.times.1005; [0362] a receptor
antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4, selected
from the group consisting of the phenothiazin-3-1s, such as
L-651,392; amidino compounds, such as CGS-25019c; benzoxalamines,
such as ontazolast; benzenecarboximidamides, such as BIIL 284/260;
and compounds, such as zafirlukast, ablukast, montelukast,
pranlukast, verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP
45715A) and BAY.times.7195; [0363] a phosphodiesterase (PDE)
inhibitor, such as a methylxanthanine, e.g. theophylline and/or
aminophylline; and/or a selective PDE isoenzyme inhibitor, e.g. a
PDE4 inhibitor and/or inhibitor of the isoform PDE4D and/or an
inhibitor of PDE5; [0364] a histamine type 1 receptor antagonist,
such as cetirizine, loratadine, desloratadine, fexofenadine,
acrivastine, terfenadine, astemizole, azelastine, levocabastine,
chlorpheniramine, promethazine, cyclizine, and/or mizolastine
(generally applied orally, topically or parenterally); [0365] a
proton pump inhibitor (such as omeprazole) or gastroprotective
histamine type 2 receptor antagonist; [0366] an antagonist of the
histamine type 4 receptor; [0367] an alpha-1/alpha-2 adrenoceptor
agonist vasoconstrictor sympathomimetic agent, such as
propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine,
pseudoephedrine, naphazoline hydrochloride, oxymetazoline
hydrochloride, tetrahydrozoline hydrochloride, xylometazoline
hydrochloride, tramazoline hydrochloride and ethylnorepinephrine
hydrochloride; [0368] an anticholinergic agent, e.g. a muscarinic
receptor (M1, M2, and M3) antagonist, such as atropine, hyoscine,
glycopyrrrolate, ipratropium bromide, tiotropium bromide,
oxitropium bromide, pirenzepine and telenzepine; [0369] a
beta-adrenoceptor agonist (including beta receptor subtypes 1-4),
such as isoprenaline, salbutamol, formoterol, salmeterol,
terbutaline, orciprenaline, bitolterol mesylate and/or pirbuterol,
e.g. a chiral enantiomer thereof; [0370] a chromone, e.g. sodium
cromoglycate and/or nedocromil sodium; [0371] a glucocorticoid,
such as flunisolide, triamcinolone acetonide, beclomethasone
dipropionate, budesonide, fluticasone propionate, ciclesonide,
and/or mometasone furoate; [0372] an agent that modulate nuclear
hormone receptors, such as a PPAR; [0373] an immunoglobulin (Ig) or
Ig preparation or an antagonist or antibody modulating Ig function,
such as anti-IgE (e.g. omalizumab); [0374] other systemic or
topically-applied anti-inflammatory agent, e.g. thalidomide or a
derivative thereof, a retinoid, dithranol and/or calcipotriol;
[0375] combinations of aminosalicylates and sulfapyridine, such as
sulfasalazine, mesalazine, balsalazide, and olsalazine; and
immunomodulatory agents, such as the thiopurines; and
corticosteroids, such as budesonide; [0376] an antibacterial agent,
e.g. a penicillin derivative, a tetracycline, a macrolide, a
beta-lactam, a fluoroquinolone, metronidazole and/or an inhaled
aminoglycoside; and/or an antiviral agent, e.g. acyclovir,
famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine,
rimantadine; ribavirin; zanamavir and/or oseltamavir; a protease
inhibitor, such as indinavir, nelfinavir, ritonavir and/or
saquinavir; a nucleoside reverse transcriptase inhibitor, such as
didanosine, lamivudine, stavudine, zalcitabine, zidovudine; a
non-nucleoside reverse transcriptase inhibitor, such as nevirapine,
efavirenz; [0377] a cardiovascular agent, such as a calcium channel
blocker, beta-adrenoceptor blocker, angiotensin-converting enzyme
(ACE) inhibitor, angiotensin-2 receptor antagonist; lipid lowering
agent, such as a statin and/or fibrate; a modulator of blood cell
morphology, such as pentoxyfylline; a thrombolytic and/or an
anticoagulant, e.g. a platelet aggregation inhibitor; [0378] a CNS
agent, such as an antidepressant (such as sertraline),
anti-Parkinsonian drug (such as deprenyl, L-dopa, ropinirole,
pramipexole; MACS inhibitor, such as selegine and rasagiline; comP
inhibitor, such as tasmar; A-2 inhibitor, dopamine reuptake
inhibitor, NMDA antagonist, nicotine agonist, dopamine agonist
and/or inhibitor of neuronal nitric oxide synthase) and an
anti-Alzheimer's drug, such as donepezil, rivastigmine, tacrine,
COX-2 inhibitor, propentofylline or metrifonate; [0379] an agent
for the treatment of acute and chronic pain, e.g. a centrally or
peripherally-acting analgesic, such as an opioid analogue or
derivative, carbamazepine, phenytoin, sodium valproate,
amitryptiline or other antidepressant agent, paracetamol, or
non-steroidal anti-inflammatory agent; [0380] a parenterally or
topically-applied (including inhaled) local anaesthetic agent, such
as lignocaine or an analogue thereof; [0381] an anti-osteoporosis
agent, e.g. a hormonal agent, such as raloxifene, or a
biphosphonate, such as alendronate; [0382] (i) a tryptase
inhibitor; (ii) a platelet activating factor (PAF) antagonist;
(iii) an interleukin converting enzyme (ICE) inhibitor; (iv) an
IMPDH inhibitor; (v) an adhesion molecule inhibitors including
VLA-4 antagonist; (vi) a cathepsin; (vii) a kinase inhibitor, e.g.
an inhibitor of tyrosine kinases (such as Btk, Itk, Jak3 MAP
examples of inhibitors might include Gefitinib, Imatinib mesylate),
a serine/threonine kinase (e.g. an inhibitor of MAP kinase, such as
p38, JNK, protein kinases A, B and C and IKK), or a kinase involved
in cell cycle regulation (e.g. a cylin dependent kinase); (viii) a
glucose-6 phosphate dehydrogenase inhibitor; (ix) a kinin-B.sub1.-
and/or B.sub2.-receptor antagonist; (x) an anti-gout agent, e.g.
colchicine; (xi) a xanthine oxidase inhibitor, e.g. allopurinol;
(xii) a uricosuric agent, e.g. probenecid, sulfinpyrazone, and/or
benzbromarone; (xiii) a growth hormone secretagogue; (xiv)
transforming growth factor (TGF(3); (xv) platelet-derived growth
factor (PDGF); (xvi) fibroblast growth factor, e.g. basic
fibroblast growth factor (bFGF); (xvii) granulocyte macrophage
colony stimulating factor (GM-CSF); (xviii) capsaicin cream; (xix)
a tachykinin NK.sub1. and/or NK.sub3. receptor antagonist, such as
NKP-608C, SB-233412 (talnetant) and/or D-4418; (xx) an elastase
inhibitor, e.g. UT-77 and/or ZD-0892; (xxi) a TNF-alpha converting
enzyme inhibitor (TACE); (xxii) induced nitric oxide synthase
(iNOS) inhibitor or (xxiii) a chemoattractant receptor-homologous
molecule expressed on TH2 cells (such as a CRTH2 antagonist);
(xxiv) an inhibitor of a P38 (xxv) agent modulating the function of
Toll-like receptors (TLR) and (xxvi) an agent modulating the
activity of purinergic receptors, such as P2X7; (xxvii) an
inhibitor of transcription factor activation, such as NFkB, API,
and/or STATS.
[0383] An inhibitor may be specific or may be a mixed inhibitor,
e.g. an inhibitor targeting more than one of the molecules (e.g.
receptors) or molecular classes mentioned above.
[0384] The binding member could also be used in association with a
chemotherapeutic agent or another tyrosine kinase inhibitor in
co-administration or in the form of an immunoconjugate. Fragments
of said antibody could also be use in bispecific antibodies
obtained by recombinant mechanisms or biochemical coupling and then
associating the specificity of the above described antibody with
the specificity of other antibodies able to recognize other
molecules involved in the activity for which IL-6 is
associated.
[0385] For treatment of an inflammatory disease, a binding member
of the invention may be combined with one or more agents, such as
non-steroidal anti-inflammatory agents (hereinafter NSAIDs)
including non-selective cyclo-oxygenase (COX)-1/COX-2 inhibitors
whether applied topically or systemically, such as piroxicam,
diclofenac, propionic acids, such as naproxen, flurbiprofen,
fenoprofen, ketoprofen and ibuprofen, fenamates, such as mefenamic
acid, indomethacin, sulindac, azapropazone, pyrazolones, such as
phenylbutazone, salicylates, such as aspirin); selective COX-2
inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib,
lumarocoxib, parecoxib and etoricoxib); cyclo-oxygenase inhibiting
nitric oxide donors (CINODs); glucocorticosteroids (whether
administered by topical, oral, intra-muscular, intra-venous or
intra-articular routes); methotrexate, leflunomide;
hydroxychloroquine, d-penicillamine, auranofin or other parenteral
or oral gold preparations; analgesics; diacerein; intra-articular
therapies, such as hyaluronic acid derivatives; and nutritional
supplements, such as glucosamine.
[0386] A binding member of the invention can also be used in
combination with an existing therapeutic agent for the treatment of
cancer. Suitable agents to be used in combination include: [0387]
(i) antiproliferative/antineoplastic drugs and combinations
thereof, as used in medical oncology, such as Gleevec (imatinib
mesylate), alkylating agents (for example cis-platin, carboplatin,
cyclophosphamide, nitrogen mustard, melphalan, chlorambucil,
busulphan and nitrosoureas); antimetabolites (for example
antifolates, such as fluoropyrimidines like 5-fluorouracil and
tegafur, raltitrexed, methotrexate, cytosine arabinoside,
hydroxyurea, gemcitabine and paclitaxel); antitumour antibiotics
(for example anthracyclines like adriamycin, bleomycin,
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin and mithramycin); antimitotic agents (for example
vinca alkaloids like vincristine, vinblastine, vindesine and
vinorelbine and taxoids like taxol and taxotere); and topoisomerase
inhibitors (for example epipodophyllotoxins like etoposide and
teniposide, amsacrine, topotecan and camptothecins); (ii)
cytostatic agents, such as antioestrogens (for example tamoxifen,
toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen
receptor down regulators (for example fulvestrant), antiandrogens
(for example bicalutamide, flutamide, nilutamide and cyproterone
acetate), LHRH antagonists or LHRH agonists (for example goserelin,
leuprorelin and buserelin), progestogens (for example megestrol
acetate), aromatase inhibitors (for example as anastrozole,
letrozole, vorazole and exemestane) and inhibitors of
5.alpha.-reductase, such as finasteride; [0388] (iii) Agents which
inhibit cancer cell invasion (for example metalloproteinase
inhibitors like marimastat and inhibitors of urokinase plasminogen
activator receptor function); [0389] (iv) inhibitors of growth
factor function, for example such inhibitors include growth factor
antibodies, growth factor receptor antibodies (for example the
anti-erbb2 antibody trastuzumab and the anti-erbb1 antibody
cetuximab [C225]), farnesyl transferase inhibitors, tyrosine kinase
inhibitors and serine/threonine kinase inhibitors, for example
inhibitors of the epidermal growth factor family (for example EGFR
family tyrosine kinase inhibitors, such as
N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)
quinazolin-4-amine (gefitinib, AZD1839),
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine
(erlotinib, OSI-774) and
6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoli-
n-4-amine (CI 1033)), for example inhibitors of the
platelet-derived growth factor family and for example inhibitors of
the hepatocyte growth factor family; [0390] (v) antiangiogenic
agents, such as those which inhibit the effects of vascular
endothelial growth factor (for example the anti-vascular
endothelial cell growth factor antibody bevacizumab, compounds,
such as those disclosed in International Patent Applications WO
97/22596, WO 97/30035, WO 97/32856 and WO 98/13354, each of which
is incorporated herein in its entirety) and compounds that work by
other mechanisms (for example linomide, inhibitors of integrin
.alpha.v.beta.3 function and angiostatin); [0391] (vi) vascular
damaging agents, such as combretastatin A4 and compounds disclosed
in International Patent Applications WO 99/02166, WO 00/40529, WO
00/41669, WO 01/92224, WO 02/04434 and WO 02/08213 (each of which
is incorporated herein in its entirety); [0392] (vii) antisense
therapies, for example those which are directed to the targets
listed above, such as ISIS 2503, an anti-ras antisense; [0393]
(viii) gene therapy approaches, including for example approaches to
replace aberrant genes, such as aberrant p53 or aberrant BRCA1 or
BRCA2, GDEPT (gene directed enzyme pro-drug therapy) approaches,
such as those using cytosine deaminase, thymidine kinase or a
bacterial nitroreductase enzyme and approaches to increase patient
tolerance to chemotherapy or radiotherapy, such as multi-drug
resistance gene therapy; and [0394] (ix) immunotherapeutic
approaches, including for example ex vivo and in vivo approaches to
increase the immunogenicity of patient tumour cells, such as
transfection with cytokines, such as interleukin 2, interleukin 4
or granulocyte macrophage colony stimulating factor, approaches to
decrease T-cell anergy, approaches using transfected immune cells,
such as cytokine-transfected dendritic cells, approaches using
cytokine-transfected tumour cell lines and approaches using
anti-idiotypic antibodies.
[0395] A binding member of the invention and one or more of the
above additional medicinal components may be used in the
manufacture of a medicament. The medicament may be for separate or
combined administration to an individual, and accordingly may
comprise the binding member and the additional component as a
combined preparation or as separate preparations. Separate
preparations may be used to facilitate separate and sequential or
simultaneous administration, and allow administration of the
components by different routes e.g. oral and parenteral
administration.
[0396] In accordance with the present invention, compositions
provided may be administered to mammals. Administration is normally
in a "therapeutically effective amount", this being sufficient to
show benefit to a patient. Such benefit may be at least
amelioration of at least one symptom. The actual amount
administered, and rate and time-course of administration, will
depend on the nature and severity of what is being treated, the
particular mammal being treated, the clinical condition of the
individual patient, the cause of the disorder, the site of delivery
of the composition, the type of binding member, the method of
administration, the scheduling of administration and other factors
known to medical practitioners. Prescription of treatment, e.g.
decisions on dosage etc, is within the responsibility of general
practitioners and other medical doctors and may depend on the
severity of the symptoms and/or progression of a disease being
treated. Appropriate doses of antibody are well known in the art
[110, 111]. Specific dosages indicated herein or in the Physician's
Desk Reference (2003) as appropriate for the type of medicament
being administered may be used. A therapeutically effective amount
or suitable dose of a binding member of the invention can be
determined by comparing its in vitro activity and in vivo activity
in an animal model. Methods for extrapolation of effective dosages
in mice and other test animals to humans are known. The precise
dose will depend upon a number of factors, including whether the
antibody is for diagnosis, prevention or for treatment, the size
and location of the area to be treated, the precise nature of the
antibody (e.g. whole antibody, fragment or diabody) and the nature
of any detectable label or other molecule attached to the antibody.
A typical antibody dose will be in the range 100 .mu.g to 1 g for
systemic applications, and 1 .mu.g to 1 mg for topical
applications. An initial higher loading dose, followed by one or
more lower doses, may be administered. Typically, the antibody will
be a whole antibody, e.g. the IgG1 isotype. This is a dose for a
single treatment of an adult patient, which may be proportionally
adjusted for children and infants, and also adjusted for other
antibody formats in proportion to molecular weight. Treatments may
be repeated at daily, twice-weekly, weekly or monthly intervals, at
the discretion of the physician. Treatments may be every two to
four weeks for subcutaneous administration and every four to eight
weeks for intra-venous administration. Treatment may be periodic,
and the period between administrations is about two weeks or more,
e.g. about three weeks or more, about four weeks or more, or about
once a month. Treatment may be given before, and/or after surgery,
and/or may be administered or applied directly at the anatomical
site of surgical treatment.
[0397] IL-6 binding members of the invention may offer advantages
in terms of dosage and administration requirements, compared with
antibodies to sIL-6Ra. As noted elsewhere herein, circulating
levels of IL-6 are significantly lower than circulating levels of
sIL-6Ra in disease. Accordingly, use of an IL-6 binding member, as
opposed to an anti-IL-6R binding member, has significant advantages
in that the amount of drug to be manufactured for each dose to
patients may be lower. Also if the dose of an anti-IL6 therapeutic
is lower there may be significant advantages in that the low dose
facilitates sub-cutaneous injections as well as intra-venous (i.v.)
injections. It is well known to those skilled in the art that
sub-cutaneous dosing may be limited by the amount of binding
member, e.g. antibody molecule, required per dose. This is due to
the sub-cutaneous injections being limited by the volume that can
be injected at one site in the skin. Subcutaneous injection volumes
of 1.2 ml or less are typically utilised. As it may be increasingly
difficult to formulate a binding member for sub-cutaneous injection
at concentrations greater than 50 mg/ml, doses above 100 mg via
this route usually require multiple injections and more discomfort
for the patient.
[0398] Having a lower dose anti-IL-6 therapeutic may also require a
lower "loading" dose of antibody to inhibit all the systemic IL-6
compared with the systemic sIL-6Ra as this is at higher
concentrations.
[0399] Further benefits may be associated with targeting IL-6
rather than 5 IL-6 receptor, representing additional advantages of
binding members of the invention as compared with binding members
for IL-6Ra.
[0400] For example, there are literature reports which show that
the circulating levels of IL-6 are significantly lower than
circulating levels of sIL-6Ra in disease [112, 113]. As the levels
of sIL-6R are significantly higher than IL-6 levels, more
anti-sIL-6R binding member may be required to neutralise the
sIL-6Ra, compared with the amount of anti-IL-6 binding member
required to neutralise IL-6. Hence, a lower dose of an anti-ligand
binding member may be needed, compared with if an anti-receptor
binding member were used.
[0401] Targeting IL-6 ligand rather than IL-6 receptor may reduce
levels of IL-6 in disease but still allow IL-6 levels to increase
during infection, where IL-6 is up-regulated as part of the immune
response.
[0402] Kawano et al. [4] showed that IL-6 was a potent growth
factor and showed that myeloma cells freshly isolated from patients
produced IL-6 and express its receptors. Moreover, anti-IL-6
antibody inhibits the in vitro growth of myeloma cells. This is
direct evidence that an autocrine loop is operating in oncogenesis
of human myelomas. Subsequent to that study, Van Zaanen et al. [5]
demonstrated that the production of IL-6 in multiple myeloma
patients decreases when treated with an anti-IL-6 ligand
antibody.
[0403] A number of further studies show that IL-6 is involved in an
autocrine feedback loop in other cell types e.g. smooth muscle
cells (SMC) [114], U373-MG astroglioma cells [115], 3T3 adipocytes
[116], neurons [117], endothelial cells [118] and Kaposi's sarcoma
cells [119]. Inhibition of IL-6 using an anti-IL6 binding member in
disease can therefore lead to a decrease in the basal disease
production of IL-6.
[0404] Further, anti-IL-6 binding members bind IL-6 in the systemic
circulation, in contrast with binding members to IL-6 receptor
which need to penetrate the tissue in order to occupy the receptor
on the surface of cells involved in the pathology of the disease to
be treated.
[0405] Binding members to IL-6 may form an equilibrium with IL-6 in
the systemic circulation, having the effect of causing gradients
across barriers e.g. the synovial membrane, which has the net
effect of removing active IL-6 from the joint and forming an
inactive complex with the binding member. The consequence of this
is that an IL-6 binding member may have quicker onset and dosing
regime may be different and potentially easier to optimise,
compared with an IL-6R binding member.
[0406] IL-6 signalling is mediated by IL-6 binding to IL-6R and
that complex binding to gp130. Given that IL-6 and IL-6Ra binding
is of nanomolar affinity (about 5 nM) and that IL6:1L6R complex and
gp130 binding is of picomolar affinity, a binding member which
targets IL-6 faces a lower amount of competition for IL-6 binding
and so may suppress a greater proportion of IL-6 signalling.
Although this may also apply for a binding member targeting the
soluble IL-6Ra and preventing IL-6:IL-6Ra complex formation, if the
IL-6Ra is membrane bound then because of steric constraints it may
be more difficult for an anti-IL-6Ra to bind and inhibit the IL-6Ra
presented on the membrane.
EXAMPLES
Example 1. Lead Isolation
1.1 Selections
[0407] Naive human single chain Fv (scFv) phage display libraries
cloned 5 in to a phagemid vector based on the filamentous phage M13
were used for selections [120, 121]). Anti-IL-6 specific scFv
antibodies were isolated from the phage display libraries using a
series of selection cycles on recombinant human IL-6 essentially as
previously described by Vaughan et al [120] and Hawkins et al
[122]. In brief, for bio-panning selections, human IL-6 in PBS
(Dulbecco's PBS, pH7.4) was adsorbed onto wells of a microtitre
plate overnight at 4.degree. C. Wells were washed with PBS then
blocked for 1 h with PBS-Marvel (3% w/v). Purified phage in
PBS-Marvel (3% w/v) were added to the wells and allowed to bind
coated antigen for 1 h. Unbound phage was removed by a series of
wash cycles using PBS-Tween (0.1% v/v) and PBS. Bound phage
particles were eluted, infected into bacteria and rescued for the
next round of selection [120].
1.2 Inhibition of IL-6 Binding to IL-6 Receptor by Crude scFv
[0408] A representative number of individual clones from the second
round of selections were grown up in 96-well plates. ScFvs were
expressed in the bacterial periplasm and screened for their
inhibitory activity in a HTRF.RTM. (Homogeneous Time-Resolved
Fluorescence, CIS Bio international) human IL-6/human IL-6
receptor-binding assay. In this assay, samples competed for binding
to cryptate labelled human IL-6 (R&D Systems), with
biotinylated IL-6R (Peprotech). A reference anti-IL-6 mAb
(Biosource AHC0562) was included in all potency assays as a
positive control. The detailed assay method is provided in the
Materials and Methods section.
1.3 Reformatting of scFv to IgG1
[0409] Clones were converted from scFv to IgG format by sub-cloning
the VH and VL domains into vectors expressing whole antibody heavy
and light chains respectively. The VH domain was cloned into a
vector (pEU15.1) containing the human heavy chain constant domains
and regulatory elements to express whole IgG heavy chain in
mammalian cells. Similarly, the VL domain was cloned into either
vector pEU3.4 for the expression of the human kappa light chain or
pEU4.4 for the expression of the human lambda light chain constant
domains, with regulatory elements to express whole IgG light chain
in mammalian cells. Vectors for the expression of heavy chains and
light chains were originally described in ref. [123]. Cambridge
Antibody Technology vectors have been engineered simply by
introducing an OriP element. To obtain IgGs, the heavy and light
chain IgG expressing vectors were transfected into EBNA-HEK293
mammalian cells. IgGs were expressed and secreted into the medium.
Harvests were pooled and filtered prior to purification. The IgG
was purified using Protein A chromatography. Culture supernatants
are loaded on a column of appropriate size of Ceramic Protein A
(BioSepra) and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl.
Bound IgG was eluted from the column using 0.1 M Sodium Citrate (pH
3.0) and neutralised by the addition of Tris-HCl (pH 9.0). The
eluted material was buffer exchanged into PBS using Nap1O columns
(Amersham, #17-0854-02) and the concentration of IgG was determined
spectrophotometrically using an extinction coefficient based on the
amino acid sequence of the IgG [124]. The purified IgG were
analysed for aggregation or degradation using SEC-HPLC and by
SDS-PAGE.
1.4 Inhibition of IL-6 Binding to IL-6 Receptor by Purified scFv 30
and IgG
[0410] ScFv which showed a significant inhibitory effect on the
IL-6:IL-6R interaction as crude periplasmic extracts, were
subjected to DNA sequencing [120, 125]. Unique scFvs were expressed
again in bacteria and purified by affinity chromatography (as
described by Bannister et al [126]. Purified IgG samples of these
clones were also prepared as described in section 1.3. The
potencies of these samples were determined by competing a dilution
series of the purified preparation against biotinylated sIL-6R for
binding to HIS FLAG tagged human IL-6 (in house E. coli
derived).
[0411] The results for clone CAN022D10, as an scFv and as an IgG
having a human heavy chain and kappa light chain constant domain,
are given in Table 1. Detailed protocols are provided in Materials
and Methods section.
TABLE-US-00001 TABLE 1 Potency of CAN022D10 scFv and IgG in the
receptor-ligand HTRF biochemical assay CLONE IC50 scFv (nM) IC50
IgG (AM) CAN022D10 45 0.31
1.5 Inhibition of IL-6 Induced Proliferation of TF-1 Cells by
Purified scFv. and IgG
[0412] The neutralisation potency of purified scFv preparations
against human and cynomolgus IL-6 bioactivity was assessed using
TF-1 cell proliferation assay. TF-1 is a human premyeloid cell line
established from a patient with erythroleukaemia [134]. The TF-1
cell line is factor dependent for survival and proliferation. TF-1
cells were shown to respond to both human and cynomolgus IL-6
(in-house, E. coli derived) and were maintained in media containing
human GM-CSF (4 ng/ml, R&D Systems). Inhibition of IL-6
dependent proliferation was determined by measuring the reduction
in incorporation of tritiated thymidine into the newly synthesized
DNA of dividing cells. A detailed description of the protocol is
provided in the Materials and Methods section.
[0413] Purified scFv preparations of CAN022D10 were capable of
inhibiting the IL-6 induced proliferation of the TF-1 cells at the
maximum concentration tested, although complete inhibition was not
observed. It was therefore not possible to calculate accurate
IC.sub.50 potency data from the results obtained. When tested as a
purified IgG, the IC.sub.50 for CAN022D10 was calculated as being
93 nM.
1.6 Selectivity and Species Cross Reactivity of Antibodies in 5
DELFIA0 Epitope Competition Assays
[0414] The species cross reactivity and selectivity of antibodies
to IL-6 family members was established using DELFIA.RTM. epitope
competition assays, by measuring inhibition of biotinylated HIS
FLAG IL-6 (in-house, E. coli derived), binding each immobilised
anti-IL-6 antibody.
[0415] Titrations of purified, leukaemia inhibitory factor (LIF)
(Chemicon), ciliary neurotrophic factor (CNTF), IL-11 and
oncostatin M (all R & D Systems) were tested in each assay to
establish the potency for each structurally related protein, as
measured by IC.sub.50 values in the assay.
[0416] Titrations of IL-6 species including cynomolgus (in house E.
Coli derived), human HIS FLAG IL-6 (in house HEK-EBNA derived), rat
and murine IL-6 (both R & D Systems) were tested in each assay
to establish the species cross-reactivity of the antibodies.
Example results of this experiment are provided in Table 2. Details
of the protocol are provided in the Materials and Methods
section.
TABLE-US-00002 TABLE 2 Potencies of IL-6 related proteins and
different IL-6 species in the CAN22D10 competition assay Protein
IC.sub.50 (nM) Human IL-6 32* Cynomolgus IL-6 100* Murine IL-6 No
inhibition Rat IL-6 No inhibition Human IL-11 No inhibition Human
CNTF No inhibition Human LIF No inhibition Human Oncostatin M No
inhibition
*Values are approximations as incomplete curves were obtained for
the samples 1.7 Inhibition of Endogenous IL-6 Induced VEGF Release
from Human Synovial Fibroblast by Purified IgG
[0417] Potencies of the antibodies were evaluated for inhibition of
IL-6 induced VEGF release from human synovial fibroblasts explanted
from donors with rheumatoid arthritis. A detailed protocol for this
procedure is provided in Materials and Methods. In brief,
titrations of the test IgG were added cultured fibroblasts, which
were then stimulated by the addition of human IL-.beta. and soluble
human IL-6R.alpha. to induce IL-6 expression and enable signalling
of the cells to induce VEGF expression. Following a 48 h
incubation, supernatants were removed and tested by ELISA for the
expression of VEGF using a commercially available kit (R & D
Systems). These data were used to determine IC.sub.50 for the
CAN022D10, which was calculated as being 45 nM.
Example 2 Antibody Optimisation
[0418] 2.1 Identification of Amino Acids that May Improve Binding
of the Lead Antibody to IL-6
[0419] A strategy to identify key residues in the parent antibody
sequence that may improve binding to IL-6 was carried out by
introducing random mutations throughout the CAN022D10 scFv
sequence. This was achieved by two rounds of mutagenesis using A
Diversify.TM. PCR random mutagenesis kit (BD biosciences),
following the manufacturers instructions to incorporate on average,
8.1 mutations per kilobase in the nucleic acid sequence per round
of mutagenesis. The selections were performed essentially as
described previously (Hanes et al 2000; Methods in Enzymology, 328,
404-430). In brief, the random mutagenesis library of the parent
clone was transcribed in to mRNA and using a process of stalled
translation, mRNA-ribosome-scFv complexes were formed. These
complexes were incubated with bio-huIL-6, and those that bound to
the antigen were then captured on streptavidin-coated paramagnetic
beads. Non-specific ribosome complexes were washed away, and mRNA
was isolated from the bound ribosomal complexes, reverse
transcribed to cDNA and then amplified by PCR. This DNA was used
for the next round of selection and/or cloned out for screening.
The selection process was repeated in the presence of decreasing
concentrations of bio-huIL-6 (100 nM to 0.1 nM over 4 rounds). ScFv
isolated by ribosome display were cloned into the phagemid vector
pCANTAB6 by Nco1/Not1 restriction endonuclease digestion (New
England Biolabs) of the ribosome display construct, followed by
ligation in to Nco1/Not1 digested pCANTAB6 using T4 DNA ligase (New
England Biolabs) [127]. Ligated DNA was then transformed in to
chemically competent TG-1 cells, and crude scFv from individual
clones were competed against CAN022D10 IgG for binding to HIS/FLAG
IL-6 tested in a ligand-antibody biochemical assay.
2.2 Identification of Improved Clones Using an Antibody-Ligand
Biochemical Assay (Using CAN022D10 IgG)
[0420] Crude scFv preparations from a representative number of
individual clones for the round 3 and round 4 outputs were screened
for their inhibitory activity in a CAN022D10 IgG-IL-6 HTRF.RTM.
binding assay. In this assay, binding of biotinyated antibody and
FLAG-tagged IL-6 was detected using cryptate labelled anti-FLAG
monoclonal antibody and streptavidin XI,.sup.ent!.TM.. The detailed
assay method is provided in the Materials and Methods section.
[0421] ScFv that demonstrated a significant inhibitory effect were
5 sequenced and produced as purified preparations as described in
section 1.4. The IC.sub.50 value for each scFv was then calculated
from data obtained by a testing dilution series of the purified
sample in the HTRF antibody-ligand biochemical assay and TF-1
proliferation assay. The most potent clones in the TF-1
proliferation assay were converted to IgG with a heavy chain
constant domain and kappa light chain constant domain, as described
previously, and were re-tested in the TF-1 proliferation assay.
Example potency data for both purified scFv and IgG for each sample
is provided in Table 3.
TABLE-US-00003 TABLE 3 Examples of clones with improved potencies
in the ligand-antibody biochemical and TF-1 proliferation assays,
isolated from the ribosome display CAN022D10 random mutagenesis
library IC.sub.50 (pM) Biochemical Assay TF-1 Proliferation Assay
Clone scFv IgG* scFv IgG Antibody 2 35 36 9600 16 Antibody 3 22 43
7300 50 Antibody 4 24 43 13400 61 Antibody 5 65 26 12400 42
*Protocol was modified for IgG potency determination so scFv and
IgG potencies for each clone should not be directly compared. For
details of modifications, see Materials and Methods.
2.3 Optimisation of Parent Clone by Targeted Mutagenesis
[0422] Lead antibodies were optimised using a targeted mutagenesis
approach using affinity-based phage display selections. For the
targeted mutagenesis approach, large scFv-phage libraries derived
from the lead clones were created by oligonucleotide-directed
mutagenesis of the variable heavy (VH) and light (VL) chain
complementarity determining regions 3 (CDR3) using standard
molecular biology techniques [128]. The libraries were subjected to
affinity-based phage display selections in order to select variants
with higher affinity for IL-6. In consequence, these should show an
improved inhibitory activity for IL-6 binding its receptor. The
selections were performed essentially as described previously
[129]. In brief, the scFv phage particles were incubated with
recombinant biotinylated human IL-6 in solution (bio-huIL-6, in
house E. coli derived and modified in house). ScFv-phage bound to
antigen were then captured on streptavidin-coated paramagnetic
beads (Dynabeads.RTM. M 280) following the manufacturer's
recommendations. The selected scFv-phage particles were then
rescued as described previously [125], and the selection process
was repeated in the presence of decreasing concentrations of
bio-huIL-6 (50 nM to 0.1 nM over 3 rounds).
[0423] Upon completion of 3 rounds of selection, the VH and VL
randomised libraries were recombined to form a single library in
which clones contained randomly paired individually randomised VH
and VL sequences. Selections were then continued as previously
described in the presence of decreasing concentrations of
bio-huIL-6 (0.1 nM to 0.1 pM over a further 4 rounds).
2.4 Identification of Improved Clones from the Targeted Mutagenesis
Using an Antibody-Ligand Biochemical Assay (Using Antibody 5
IgG)
[0424] Crude scFv from clones isolated from the targeted
mutagenesis selection outputs were tested in an antibody-ligand
biochemical assay, essentially as described in section 2.2. For
these outputs, the biochemical assay was re-configured to use
Antibody 5 IgG. This antibody is an improved variant on CAN02210
with greater potency in the TF-1 proliferation assay. Incorporation
of this more potent IgG resulted in the assay that was able to
distinguish between clones of higher potency. The protocol for this
modified assay was as described for the original antibody-ligand
biochemical assay using CAN022D10, with the following changes.
Firstly the concentration of HIS FLAG IL-6 used was reduced from 1
nM to 0.5 nM. Secondly, the concentrations of the anti IL-6
antibody and streptavidin XLent!.TM. were increased from 1 nM and
20 nM to 16 nM and 40 nM respectively. ScFv that demonstrated a
significant inhibitory effect were sequenced and produced as
purified scFv and IgG, then tested in the TF-1 proliferation
assay.
2.5. Inhibition of IL-6 Induced Proliferation of TF-1 Cells by
Purified scFv and IgG of Optimised Clones
[0425] Potencies of the optimised clones were determined using the
IL-6 induced TF-1 proliferation assay as previously described.
Clones were tested as both purified scFv preparations and as
re-formatted IgG. Example results for both scFv and IgG are given
in Table 4.
TABLE-US-00004 TABLE 4 Example potencies of clones identified from
the targeted mutagenesis libraries when tested in the TF-1 cell
proliferation assay IC.sub.50 (pM) Clone (non-germlined) scFv IgG
Antibody 7 11 3 Antibody 8 419 48 Antibody 10 549 40 Antibody 14
448 31 Antibody 16 154 4.9 Antibody 17 38 16 Antibody 18 51 30
Antibody 19 508 68 Antibody 21 42 N.D. Antibody 22 41 N.D. Antibody
23 161 20 CNTO-328 N.D. 74 N.D. Not Determined
[0426] Clones demonstrated significant inhibitory effect, but
accurate IC.sub.50 values could not be determined from the dilution
series of purified scFv.
2.6. Germlining
[0427] The amino acid sequences of the VH and VL domains of the
optimised anti-IL-6 antibodies were aligned to the known human
germline sequences in the VBASE database [130], and the closest
germline was identified by sequence similarity. For the V.sub.H
domains of the CANDY022D10 antibody lineage the closest germline v
segment was Vh3_DP-86_(3-66) and the closest germline j segment was
JH2. For the VL domains the closest germline v segment was Vkl_L12
and closest germline j segment was JK2.
[0428] Without considering the Vernier residues [131], which were
left unchanged, there were 3 changes in the frameworks of the
V.sub.H domains and 4 changes in the V.sub.L domains, all of which
were reverted to the closest germline sequence to identically match
human antibodies using standard site directed mutagenesis
techniques with the appropriate mutagenic primers.
[0429] A total of 5 Vernier residues were identified in the scFv
sequence of CAN022D10 that were mutated from germline. These were
in the heavy chain at Kabat residues 29 (I present instead of V),
69 (M instead of I), 73 (I instead of N) and 78 (V instead of L). A
single Vernier mutation was also identified in the light chain
sequence at Kabat residue 46 (V instead of L).
[0430] Germlined IgG were then re-evaluated in the IL-6 induced
TF-1 proliferation assay to confirm there had not been a reduction
in potency. Example potencies for germlined (GL) antibodies are
provided in Table 5.
TABLE-US-00005 TABLE 5 Example potency data for germlined optimised
clones when evaluated in the IL-6 induced TF-1 cell proliferation
assay Clone IC.sub.50 (pM) Antibody 7 (GL) 5 Antibody 10 (GL) 71
Antibody 17 (GL) 1 Antibody 18 (GL) 3 CNTO-328 101
2.7. Inhibition of Endogenous IL-6 Induced VEGF Release from Human
5 Synovial Fibroblast by Optimised IgG
[0431] Optimised IgG were tested in the synovial fibroblast VEGF
release assay to evaluate potency against endogenously expressed
IL-6. This procedure is reviewed in section 1.7 and described in
detail in the Materials and Methods section. Example potencies for
the IgG tested are given in Table 6a. Mean potency data for the IgG
tested are given in Table 6b.
TABLE-US-00006 TABLE 6a Example potency data for optimised clones
when evaluated against endogenous IL-6 in the IL-6 induced synovial
fibroblast VEGF release assay Clone (GL = germlined clones)
IC.sub.50 (nM) Antibody 2 0.59 Antibody 3 0.38 Antibody 4 0.52
Antibody 5 0.70 Antibody 7 (GL) 0.75 Antibody 10 (GL) 0.55 Antibody
17 (GL) 0.57 Antibody 18 (GL) 0.93 CNTO-328 1.31
TABLE-US-00007 TABLE 6b Mean potency data for optimised clones when
evaluated against endogenous IL-6 in the IL-6 induced synovial
fibroblast VEGF release assay Clone (GL = germlined clones)
IC.sub.50 (nM) (95% CI) n Antibody 7 (GL) 0.78 (0.54-1.11) 3
Antibody 17 (GL) 0.57 (0.51-0.64) 3 Antibody 18 (GL) 0.67
(0.20-2.25) 4 CNTO-328 1.02 (0.39-2.63) 4
2.8. Selectivity and Species Cross Reactivity of Optimised
Antibodies in DELFIA Epitope Competition Assays
[0432] Selectivity and species cross reactivity was reevaluated for
a panel of clones using the DELFIA.RTM. epitope competition assay
as previously described (see section 1.6 and Materials and
Methods). Human and cynomolgus IL-6 produced overlapping inhibition
curves and therefore equivocal IC.sub.50 values for all IgG tested.
No inhibition was observed for murine, rat or dog IL-6 or any of
the related human proteins tested against the antibody panel. This
data demonstrates that the panel of clones tested are cross
reactive to cynomolgus IL-6 but do not bind to murine, rat or dog
IL-6, or to the most related human proteins to human IL-6.
2.9 Calculation of Affinity Data for Optimised Clones Using
BIAcore
[0433] The binding affinity of purified IgG samples of
representative antibodies 7 and 18 to human and cynomolgus IL-6
were determined by surface plasmon resonance using a BIAcore 2000
biosensor (BIAcore AB) essentially as described in ref [132]. In
brief, purified antibodies were coupled to the surface of a CM5
sensor chip using an amine coupling kit (BIAcore) to provide a
surface density of between 220-225 Ru. Human and cynomolgus IL-6 at
a range of concentrations between 200 nM and 0.2 nM in HBS-EP
buffer were passed over the sensor chip surface. The resulting
sensorgrams were evaluated using BIA evaluation 3.1 software to
provide relative binding data.
[0434] The lower limit of affinity measurement range of the BIAcore
2000.TM. biosensor is approximately 10 pM (BIAcore 2000 Instrument
handbook). From the data obtained, the affinity of the antibodies
to both human and cynomolgus IL-6 was below this 10 pM limit, i.e.
the antibodies were more potent than could be measured. Accurate
affinity measurements were therefore not calculated. The affinities
of both antibodies to both IL-6 species using this approach are
considered to be less than 10 pM.
2.10 Calculation of Affinity Data for an Optimised Clone Using the
TF-1 Cell Proliferation Assay In Vitro
[0435] The TF-1 assay was used to calculate the affinity of
Antibody 18 by use of Schild analysis. An IL-6 standard curve
(7.7.times.10.sup.-15 M to 3.times.10.sup.-9 M) was mixed with a
range of IgG concentrations (2.67.times.10.sup.-13 M to
8.3.times.10.sup.-10 M) in duplicate. By plotting the Log 10
antibody concentration against the Log 10 dose ratio, the affinity
of the IgG was determined. Using this approach the affinity of
Antibody 18 (GL) to human IL-6 was calculated as being 0.40 pM (95%
CI 0.12 pM-0.69 pM, n=6).
2.11 Antagonist Potency at Human Recombinant IL-6 Using IL-6
Mediated B9 Cell Proliferation In Vitro
[0436] IL-6 induced B9 cell proliferation was assessed in the
presence of Antibody 18 and an isotype control antibody. The
effects of a range of concentrations of each antibody
(1.times.10.sup.-13 M to 1.times.10.sup.-9 M) were assessed on an
IL-6 standard curve (concentration range 1.times.10.sup.-14 M to
1.times.10.sup.-9 M). Data points were in duplicate. B9
proliferation was determined after 4 days incubation by reduction
of alamar blue (fluorescence method).
[0437] Antibody 18 was shown to inhibit IL-6 induced B9
proliferation The isotype control had no inhibitory effect. Mean
data are shown in Table 8.
TABLE-US-00008 TABLE 8 Mean Kb values for inhibition of IL-6
induces B9 proliferation Mean Kb pM (95% CI) n Antibody 18 (GL) 0.3
(0.1-0.5) 6
2.12 Antagonist Potency at Human Recombinant IL-6 Using IL-65
Mediated IgM Release from SKW6.4 Cells In Vitro
[0438] IL-6 induces IgM secretion from the human B lymphoblast cell
line SKW 6.4. SKW6.4 cells incubated with a range of IL-6
concentrations (1.times.10.sup.-13 M to 3.times.10.sup.-8.5 M) gave
an average [A]50 of 77 pM (n=3) on IgM secretion. The effect of the
anti-human IL-6 Antibodies 7, 17 and 18 and an isotype control
antibody on IL-6 induced IgM secretion was assessed by observing
the inhibition of various antibody concentrations
(1.times.10.sup.-12.5 M to 1.times.10.sup.-8 M) in the presence of
100 pM IL-6. IgM secretion was determined after 4 days by
anti-human IgM ELISA. Data points were in duplicate.
[0439] Antibodies 7, 17 and 18 inhibited IL-6 induced IgM
secretion. The isotype control had no inhibitory effect in these
assays. Mean data is shown in Table 9.
TABLE-US-00009 TABLE 9 Mean inhibition of IgM secretion from SKW6.4
cells Mean IC50 pM n Antibody 7 (GL) 2.64 3 Antibody 17 (GL) 3.21 3
Antibody 18 (GL) 2.63 3
Example 3. Epitope Mapping
3.1 Comparison of Anti-IL-6 Antibody Epitope to Known Anti-Human
IL-6 Antibodies
[0440] The epitope of Antibody 18 (GL) was compared with the
epitopes of two anti-human IL-6 antibodies B-E8 and cCLB. Both
these antibodies are known to inhibit the binding of IL-6 to IL-6Ra
and have been investigated as potential therapeutic agents [5, 31,
34, 37, 133]. To enable comparisons of the epitopes of the three
antibodies, a panel of IL-6 mutants were constructed that each
contained a single amino acid mutation compared to the wild-type
(wt) sequence. The binding of these mutants to the different
antibodies was then evaluated in biochemical competition assays.
These experiments were based on the biochemical competition assay
described in Example 1.6, with changes in the concentrations of
antibodies and IL-6 variants where required. Briefly, antibodies
were coated on to the surface of a 96-well Nunc Maxisorp
immunoassay plate at a concentration of either 2 nM (Antibody 18)
or 4 nM (B-E8 and cCLB8) in PBS and incubated overnight at
4.degree. C. After the surface of the wells was blocked using 3%
(w/v) BSA in PBS, dilutions of the inhibitors at a concentration
range of 200 nM to 10 pM mixed with biotinylated human IL-6 at a
final concentration of 0.15 nM were added to the antibody coated
wells and allowed to bind. Binding of the biotinylated IL-6 to the
antibodies was measured using Europium labelled streptavidin.
[0441] By comparing the IC.sub.50 values obtained for the mutants
to unlabeled wild type human IL-6, a ratio of potency could be
established for each mutant. Then, by comparing these ratios across
the different antibodies, the effects of the individual mutations
on the binding of the antibody to the IL-6 molecule could be
evaluated. Typical results of these experiments are presented in
Table 10 with the experiments being repeated on 2 further
occasions.
TABLE-US-00010 TABLE 10 IC50 and potency ratios of a panel of IL-6
mutants against the anti-human IL-6 antibodies antibody 18, B-E8
and cCLB8 IC50 (M) Potency Ratio Mutant Antibody 18 CNTO-328 B-E8
Antibody 18 CNTO-328 B-E8 F102E 8.41E-08 2.80E-09 1.58E-08 310.951
3.021 57.246 F106E 1.43E-09 No inhibition 3.31E-09 5.283 -- 11.989
Irrelevant No inhibition No inhibition No inhibition -- -- -- wt
IL-6 2.70E-10 9.26E-10 2.76E-10 1.000 1.000 1.000 R207E No
inhibition 5.13E-09 No inhibition -- 7.401 -- Q211A 3.07E-10
1.10E-07 8.51E-10 1.498 158.221 3.208 Irrelevant No inhibition No
inhibition No inhibition -- -- -- wt IL-6 2.05E-10 6.93E-10
2.65E-10 1.000 1.000 1.000 R58E 4.79E-10 1.73E-09 1.57E-08 2.009
1.682 79.083 S204E 2.57E-08 1.90E-09 4.83E-10 107.754 1.848 2.434
Irrelevant No inhibition No inhibition No inhibition -- -- wt IL-6
2.39E-10 1.03E-09 1.98E-10 1.000 1.000 1.000 E200W 5.22E-10
2.68E-09 5.68E-10 2.130 3.817 2.287 R207L 1.31E-07 1.51E-09
9.41E-08 534.666 2.148 378.865 wt IL-6 2.45E-10 7.02E-10 2.48E-10
1.000 1.000 1.000
The residue numbering in table 10 is for the amino acid sequence of
full length human IL-6 (SEQ ID NO: 161).
[0442] The results show that the three antibodies have different
binding profiles against the panel of IL-6 mutants and therefore
bind to different epitopes on the surface of the cytokine. Kalai et
al (1997) previously observed that cCLB8 does not recognise the
IL-6 mutant F106E. This has been confirmed in our experiments, as
it does not inhibit binding of the biotinylated IL-6 to the
antibody. In contrast, the IL-6 mutant F106E is only 5-fold less
potent than the wt IL-6 in the competition assay using Antibody 18,
indicating that it binds strongly to this antibody. A similar
result was observed with mutant Q211A, where the potency ratio
against antibody 18 was 1.5, compared to 158 for cCLB8. Conversely,
mutants F102E, R207E, R207L and 5204E were potent inhibitors in the
cCLB8 assay but were observed to be considerably less potent than
wt IL-6 in the Antibody 18 assay.
[0443] Differences in the binding of Antibody 18 and B-E8 were
observed with mutants R58E and S204E. The potency ratio for R58E
was 2.009 for Antibody 18, compared to 79.083 for B-E8, indicating
that this mutation reduces the binding of B-E8 to IL-6. The effect
of mutation 5204E appears to be specific to Antibody 18 out of the
three antibodies tested. As with cCLB8, this mutation has little
impact on the potency of IL-6 binding to B-E8, however the mutant
is over 100-fold less potent than the wild-type IL-6 in the
biochemical assay for Antibody 18.
Example 4. Administration of an Anti-IL-6 Antibody In Vivo
4.1 Effect of Administration of an Anti-IL-6 Antibody on Human
Recombinant IL-6-Induced Neutrophil and Haptoglobin Increase in
Mice
[0444] Systemic administration of IL-6 is known to cause a systemic
increase in neutrophils and acute phase protein concentrations. An
in vivo model was generated where human IL-6 was administered by
intra-peritoneal injection into male C57/B/6/J mice and
concentrations of neutrophils and the acute phase protein
haptoglobin were measured. The ability of Antibody 18 (GL)
administered by sub-cutaneous injection to inhibit the responses
was measured.
4.2 Haptoglobin Assay
[0445] Intra-peritoneal injection of human IL-6 (5.2 nmol/kg,
equivalent to 12 mg/kg, b.i.d.) for 7 days resulted in a
significant increase in the plasma haptoglobin levels from
0.02.+-.0.01 mg/mL (vehicle controls) to 1.19.+-.0.27 mg/mL in the
IL-6 treated group (T-test, P<0.01). Whilst the IgG1 isotype
control had no effect, Antibody 18 dose-dependently inhibited the
response with significant inhibition (ANOVA, P<0.01 vs IL-6
alone) being noted at doses of 10.6 nmol/kg (156 mg/kg) and above
(FIG. 1).
4.3 Neutrophil Assay
[0446] Intra-peritoneal injection of human IL-6 (5.2 nmol/kg,
equivalent to 12 mg/kg, b.i.d.) for 7 days resulted in a
significant increase in neutrophil count from 1.1.+-.0.44.times.109
cells/L (vehicle controls) to 2.47.+-.0.12.times.109 cells/L in the
IL-6 treated group (T-test, P<0.01). Whilst the IgG1 isotype
control had no effect, antibody 18 dose-dependently inhibited the
response with significant inhibition (ANOVA, P<0.01 vs IL-6
alone) being noted at doses of 1.5 nmol/kg (23 mg/kg) and
above.
[0447] These results confirm the ability of an anti-IL-6 antibody
to inhibit the systemic effects of IL-6 in vivo.
Materials and Methods
[0448] Inhibition of IL-6 Binding to IL-6 Receptor by Crude
scFv
[0449] Selection outputs were screened in receptor-ligand binding
HTRF.RTM. (Homogeneous Time-Resolved Fluorescence) assay format for
inhibition of either, cryptate labelled human IL-6 (R&D Systems
206-IL), or HIS FLAG tagged human IL-6 (in house E. coli derived)
binding biotinylated IL-6R (Peprotech 200-06 R).
[0450] Outputs during lead isolation were screened as undiluted,
crude scFv containing periplasmic extracts prepared in: 200 mM
hepes buffer pH7.4, 0.5 mM EDTA and 0.5 M sucrose. 8 nM
biotinylated human IL-6R was pre-incubated for 30 minutes at room
temperature in the dark, with 8 nM streptavidin XL.sup.ent!.TM.
(CIS Bio International 611SAXLA). All dilutions were done in
phosphate buffered saline (PBS) containing 0.4 M potassium fluoride
and 0.1% BSA (assay buffer).
[0451] After pre-incubation of the reagents, 10 .mu.l of crude scFv
sample was added to a 384 well low volume assay plate (Costar
3676). This was followed by the addition of 5 .mu.l of the
pre-incubated biotinylated receptor and streptavidin
XL.sup.ent!.TM. mix, and then 5 .mu.l of 11.2 nM cryptate labelled
human IL-6.
[0452] Assay plates were then centrifuged at 1000 rpm at room
temperature for 1 min, and incubated for 2 h at room temperature,
prior to reading time resolved fluorescence at 620 nm and 665 nm
emission wavelengths using an EnVision plate reader (Perkin
Elmer).
Inhibition of IL-6 Binding to IL-6 Receptor by Purified scFv and
IgG
[0453] Purified scFv and IgG from positive clones identified from
screening were tested in a HTRF.RTM. assay for inhibition of
binding of HIS FLAG tagged human IL-6 to biotinylated IL-6R. 8 nM
biotinylated human IL-6R was pre-incubated for 30 minutes at room
temperature in the dark, with 8 nM streptavidin XL.sup.ent!.TM..
All dilutions were done in phosphate buffered saline (PBS)
containing 0.4 M potassium fluoride and 0.1% BSA (assay
buffer).
[0454] A titration of the purified sample was used in order to
establish the clone potency as measured by IC.sub.50 values in the
assay. After pre-incubation of the reagents, 10 .mu.l of titration
of purified scFv sample was added to a 384 well low volume assay
plate (Costar 3676). This was followed by the addition of 5 .mu.l
of the pre-incubated biotinylated receptor and streptavidin
XL.sup.ent!.TM. mix. 2 nM HIS FLAG tagged human IL-6 was combined
with 1.732 nM anti-flag IgG labelled with cryptate (CIS Bio
International 61FG2KLB) and immediately 5 .mu.l of mix was added to
assay plate.
[0455] Assay plates were then centrifuged at 1000 rpm at room
temperature for 1 min, and incubated for 2 h at room temperature,
prior to reading time resolved fluorescence at 620 nm and 665 nm
emission wavelengths using an EnVision plate reader (Perkin
Elmer).
Data Analysis
[0456] The following methods were used to analyse data from the
HTRF.RTM. assays described above.
[0457] Data was analysed by calculating % Delta F values for each
sample. Delta F was determined according to equation 1.
% .times. .times. Delta .times. .times. F = ( sample .times.
.times. 665 .times. .times. nm .times. / .times. 620 .times.
.times. nm .times. .times. ratio .times. .times. value ) - ( non
.times. - .times. specific .times. .times. control .times. .times.
665 .times. .times. nm .times. / .times. 620 .times. .times. nm
.times. .times. ratio .times. .times. value ) ( non .times. -
.times. specific .times. .times. control .times. .times. 665
.times. .times. nm .times. / .times. 620 .times. .times. nm .times.
.times. ratio .times. .times. value ) .times. 100 Equation .times.
.times. 1 ##EQU00001##
% Delta F values were subsequently used to calculate % specific
binding as described in equation 2.
% .times. .times. specific .times. .times. binding = % .times.
.times. Delta .times. .times. F .times. .times. of .times. .times.
sample % .times. .times. Delta .times. .times. F .times. .times. of
.times. .times. total .times. .times. binding .times. .times.
control .times. 100 Equation .times. .times. 2 ##EQU00002##
[0458] IC.sub.50 values were determined using GraphPad Prism
software by curve fitting using a four-parameter logistic equation
(Equation 3).
Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log
EC50-X)*HillSlope)) Equation 3:
[0459] X is the logarithm of concentration. Y is specific
binding
[0460] Y starts at Bottom and goes to Top with a sigmoid shape.
[0461] A reference anti-IL-6 mAb (Biosource AHC0562) was included
in all assays as a positive control.
Inhibition of IL-6 Induced Proliferation of TF-1 Cells by Purified
scFv and IgG
[0462] TF-1 cells were a gift from R&D Systems and maintained
according to supplied protocols. Assay media comprised RPMI-1640
with GLUTAMAX I (Invitrogen) containing 5% foetal bovine serum
(JRH) and 1% sodium pyruvate (Sigma). Prior to each assay, TF-1
cells were pelleted by centrifugation at 300.times.g for 5 mins,
the media removed by aspiration and the cells re-suspended in assay
media. This process was repeated twice with cells re-suspended at a
final concentration of 5.times.10.sup.5 cells/ml in assay media.
The cells were plated out using 100 .mu.l/well in a 96 well assay
plate. Plates were incubated for 24 hours at 37.degree. C. and 5%
CO.sub.2 to starve cell of GM-CSF. Test solutions of purified scFv
or IgG (in duplicate) were diluted to the desired concentration in
assay media. An irrelevant antibody not directed at IL-6 was used
as negative control. Recombinant bacterially derived human
(R&D) and cynomolgus (in-house) IL-6 was added to a final
concentration of either 20 pM (human IL-6) or 100 pM (cynomolgus)
when mixed with appropriate test antibody in a total volume of 100
.mu.l/well. The concentration of IL-6 used in the assay was
selected as the dose that at final assay concentration gave
approximately 80% of maximal proliferative response. All samples
were incubated for 30 mins at room temperature. 100 .mu.l of IL-6
and antibody mixture was then added to 100 .mu.l of the cells to
give a total assay volume of 200 .mu.l/well. Plates were incubated
for 24 hours at 37.degree. C. and 5% CO.sub.2, 200 of tritiated
thymidine (5 .mu.Ci/ml) was then added to each assay point and the
plates were returned to the incubator for further 24 hours. Cells
were harvested on glass fibre filter plates (Perkin Elmer) using a
cell harvester. Thymidine incorporation was determined using
Packard TopCount microplate liquid scintillation counter. Data was
then analysed using Graphpad Prism software.
Method for Time Resolved Fluorescence Assay of Inhibition of
Biotinylated Human IL-6 Binding to Immobilised Anti IL-6
Antibodies
[0463] The specific method used for this assay and for which
results are provided in Example 2.6 employed DELFIA.RTM. reagents
and is set out above. The method is also described more generally
below, and is suitable as an assay for determining and/or
quantifying binding of other IL-6 forms and related proteins to
anti IL-6 MAbs.
[0464] In this assay, the anti-IL-6 monoclonal antibody is bound to
a solid support, for example being attached to the support via Fc.
Polystyrene high protein binding plates, e.g. Nunc Maxisorb plates,
may be used as a suitable support. [0465] Coat the anti IL-6 MAb on
to plates at 50 .mu.l per well in PBS, overnight at 4.degree. C.
[0466] All subsequent steps are performed at room temperature.
[0467] Wash plates three times with PBS, containing 0.05% Tween20
(PBST, currently available under Sigma P1379), then block with 300
.mu.l/well PBS containing 3% (w/v) BSA (currently available under
Roche Diagnostics, 70129138) for 1 h. [0468] Wash plates three
times with PBST. [0469] Prepare inhibitor titrations in PBS
containing 3% (w/v) BSA and add to a `dilution` plate (40
.mu.l/well) followed by 40 .mu.l/well biotinylated IL-6 to give a
final concentration of biotinylated IL-6 equivalent to the KD for
the protein for the antibody. Transfer 50 .mu.l of the samples from
the dilution plate to the corresponding wells in the assay plate
[0470] Incubate plates for 1 h. [0471] Wash plates three times with
PEST then to each well add 50 .mu.l/well of 0.1 .mu.m/ml
Europium-labelled streptavidin in 50 mM Tris-HCl, pH 7.5,
containing 0.9% NaCl, 0.5% purified BSA, 0.1% Tween20 and 20 .mu.m
EDTA and incubate for 1 h. [0472] Wash plates seven times with a
wash buffer comprising of 0.05 M Tris buffered saline (0.138 M
NaCl, 0.0027 M KCl), 0.05% (v/v) Tween20, pH8.0 (at 25.degree. C.)
[0473] To each well, add 50 .mu.l of an enhancement solution,
acidified with acetic acid and containing Triton X-100 along with
the chelators .beta.NTA and TOPO. The resulting pH shift from
alkali to acid causes a rapid dissociation of the europium ions
from the streptavidin conjugate. The free Europium ions then form
fluorogenic chelates with the available chelators. Water is removed
by the presence of TOPO, enabling the chelates to form micelles,
prolonging the fluorogenicity of the chelate. [0474] Incubate for 5
min, then measure time resolved fluorescence at a 620 nm emission
wavelength. Fluorescence data are converted to % specific binding
according to Equation 1. Determine total binding from control wells
containing biotinylated huIL-6 but no competitor. Determine
non-specific binding from wells containing biotinylated huIL-6 and
a 100-fold excess of huIL-6. Fit resultant data to a sigmoidal
curve for calculation of IC.sub.50 values according to Equation 2.
Determination of Antibody Coating and Biotinylated huIL-6
Concentrations for the Biochemical Epitope Competition Assay
[0475] The concentration of antibody used for coating and the
concentration of biotinylated huIL-6 used in the epitope
competition assay will depend on the affinity of the interaction of
the two reagents and the efficiency of antibody immobilisation. A
standard concentration for antibody coating and the concentration
of biotinylated huIL-6 required must therefore be determined for
each antibody to be tested.
[0476] As a general rule, the final concentration biotinylated
huIL-6 used in each assay is equlivalent to the KD of the ligand
for the corresponding antibody as determined by saturation
analysis. The concentration of antibody used for coating should be
such that when the biotinylated huIL-6 is added at KD a minimum
signal to background ratio of 10:1 is obtained when detected under
the competition assay conditions.
Selectivity and Species Cross Reactivity of Antibodies in
DELFIA.RTM. Epitope Competition Assays
[0477] Purified IgG were adsorbed onto 96-well Maxisorp microtitre
plates (Nunc) in PBS at a concentration which gave a significant
signal when biotinylated human IL-6 was added at approximately its
estimated Kd for that particular IgG. Excess IgG was washed away
with PBS-Tween (0.1% v/v) and the wells were blocked with
PBS-Marvel (3% w/v) for 1 h. A dilution series of each of the
following competitors was prepared in PBS, starting at a
concentration of approximately 200-times the Kd value of the
Interaction between biotinylated human IL-6 and the respective IgG;
Human IL-6, Cynomolgus IL-6, Rat IL-6 (R & D Systems
506-RL/CF), Murine IL-6 (R & D Systems 406-ML/CF), Human CNTF
(R & D Systems 257-NT/CF), Human LIF (Chemicon, LIF1010), Human
IL-11(R & D Systems 518-IL/CF) Human Oncostatin M (R & D
Systems 295-OM/CF) Unbiotinylated human IL-6 was used as a positive
control. To this series, an equal volume of biotinylated
recombinant human IL-6 at a concentration of approximately 2-fold
the Kd was added (resulting in a series starting at a ratio of
competitor antigen:biotinylated human IL-6 of approximately 100:1).
These mixtures were then transferred onto the blocked IgG and
allowed to equilibrate for 1.5 h. Unbound antigen was removed by
washing with PBS-Tween (0.1% v/v), while the remaining biotinylated
human IL-6 was detected by streptavidin-Europium3+ conjugate
(DELFIA.RTM., detection, PerkinElmer). Time-resolved fluorescence
was measured at 620 nm on an EnVision plate reader (PerkinElmer).
Fluorescence data was converted to % specific binding (100% was
determined from control wells containing biotinylated human IL-6
but no competitor, 0% was from wells containing biotinylated human
IL-6 and a 100-fold excess of unbiotinylated human IL-6). Resultant
data were analysed using Prism curve fitting software (Graphpad) to
determine IC50 values according to Equation 3.
Method for Time Resolved Fluorescence Assay of Inhibition of
Biotinylated Human IL-6 Binding to Immobilised Anti IL-6
Antibodies
[0478] The specific method used for this assay and for which
results are provided in Example 2.8 employed DELFIAC, reagents and
is set out above. The method is also described more generally
below, and is suitable as an assay for determining and/or
quantifying binding of other IL-6 forms and related proteins to
anti IL-6 MAbs.
[0479] In this assay, the anti-IL-6 monoclonal antibody is bound to
a solid support, for example being attached to the support via Fc.
Polystyrene high protein binding plates, e.g. Nunc Maxisorb plates,
may be used as a suitable support. [0480] Coat the anti IL-6 MAb on
to plates at 50 .mu.l per well in PBS, overnight at 4.degree. C.
[0481] All subsequent steps are performed at room temperature.
[0482] Wash plates three times with PBS, containing 0.05% Tween20
(PBST, currently available under Sigma P1379), then block with 300
.mu.l/well PBS containing 3% (w/v) BSA (currently available under
Roche Diagnostics, 70129138) for 1 h. [0483] Wash plates three
times with PBST. [0484] Prepare inhibitor titrations in PBS
containing 3% (w/v) BSA and add to a `dilution` plate (40
.mu.l/well) followed by 40 .mu.l/well biotinylated IL-6 to give a
final concentration of biotinylated IL-6 equivalent to the KD for
the protein for the antibody. [0485] Transfer 50 .mu.l of the
samples from the dilution plate to the corresponding wells in the
assay plate [0486] Incubate plates for 1 h. [0487] Wash plates
three times with PBST then to each well add 50 .mu.l/well of 0.1
.mu.g/ml Europium-labelled streptavidin in 50 mM Tris-HCl, pH 7.5,
containing 0.9% NaCl, 0.5% purified BSA, 0.1% Tween20 and 20 .mu.m
EDTA and incubate for 1 h. [0488] Wash plates seven times with a
wash buffer comprising of 0.05 M Tris buffered saline (0.138 M
NaCl, 0.0027 M KCl), 0.05% (v/v) Tween20, pH8.0 (at 25.degree. C.)
[0489] To each well, add 50 .mu.l of an enhancement solution,
acidified with acetic acid and containing Triton X-100 along with
the chelators .beta.NTA and TOPO. The resulting pH shift from
alkali to acid causes a rapid dissociation of the europium ions
from the streptavidin conjugate. The free Europium ions then form
fluorogenic chelates with the available chelators. Water is removed
by the presence of TOPO, enabling the chelates to form micelles,
prolonging the fluorogenicity of the chelate. [0490] Incubate for 5
min, then measure time resolved fluorescence at a 620 nm emission
wavelength. Fluorescence data are converted to % specific binding
according to Equation 1. Determine total binding from control wells
containing biotinylated huIL-6 but no competitor. Determine
non-specific binding from wells containing biotinylated huIL-6 and
a 100-fold excess of huIL-6. Fit resultant data to a sigmoidal
curve for calculation of IC50 values according to Equation 2.
Determination of Antibody Coating and Biotinylated huIL-6
Concentrations for the Biochemical Epitope Competition Assay
[0491] The concentration of antibody used for coating and the
concentration of biotinylated huIL-6 used in the epitope
competition assay will depend on the affinity of the interaction of
the two reagents and the efficiency of antibody immobilisation. A
standard concentration for antibody coating and the concentration
of biotinylated huIL-6 required must therefore be determined for
each antibody to be tested.
[0492] As a general rule, the final concentration biotinylated
huIL-65 used in each assay is equlivalent to the KD of the ligand
for the corresponding antibody as determined by saturation
analysis. The concentration of antibody used for coating should be
such that when the biotinylated huIL-6 is added at KD a minimum
signal to background ratio of 10:1 is obtained when detected under
the competition assay conditions.
Identification of Improved Clones Using an Antibody-Ligand
Biochemical Assay
[0493] Selection outputs from lead optimisation were screened in
epitope competition HTRF.RTM. assay format for inhibition of HIS
FLAG tagged human IL-6 (in house E. coli derived) binding
biotinylated anti IL-6 antibody (in house IgG derived from lead
isolation, CAN022D10).
[0494] Outputs during lead optimisation were screened as undiluted,
crude scFv containing periplasmic extracts prepared in; 50 nM MOPS
buffer pH7.4, 0.5 mM EDTA and 0.5 M Sorbitol. 1 nM human HIS FLAG
IL-6 was pre-incubated for 30 minutes at room temperature in the
dark, with 1.732 nM anti-flag IgG labelled with cryptate (CIS Bio
International 61FG2KLB). All dilutions were performed in assay
buffer. In parallel, 1 nM of biotinylated anti-IL-6 IgG (against
which competition of a test binding member was to be tested) was
pre-incubated for 30 minutes at room temperature in the dark with
20 nM of streptavidin XL.sup.ent!.TM. (CIS Bio International
611SAXLB).
[0495] After pre-incubation of reagents, 10 pl of crude scFv sample
was added to a black 384 well optiplate (Perkin Elmer Cat No.
6007279). This was followed by addition of 10 .mu.l assay buffer to
the whole plate. Then 10 .mu.l of the pre-incubated biotinylated
anti-IL-6 IgG and Streptavidin XLentl.TM. mix, and 10 .mu.l of
pre-incubated HIS FLAG tagged human IL-6 anti-flag cryptate mix
were added.
[0496] Assay plates were then centrifuged at 1000 rpm at room
temperature for 1 min, and incubated for 2 h at room temperature,
prior to reading time resolved fluorescence at 620 nm and 665 nm
emission wavelengths using an EnVision plate reader (Perkin Elmer).
Data was analysed by calculating % deltaF and % specific binding as
previously described.
[0497] Following identification of improved leads from the random
mutagenesis library, undiluted crude scFv outputs from CDR3
targeted mutagenesis selections were screened in a modified version
of the epitope competition HTRF.RTM. assay which included the
following changes 0.5 nM human HIS FLAG IL-6 was pre-incubated for
30 minutes at room temperature in the dark, with 1.732 nM anti-flag
IgG labelled with cryptate (CIS Bio International 61FG2KLB). In
parallel, 16 nM of biotinylated anti-IL-6 IgG (Antibody 5, in house
IgG identified from CAN022D10 random mutagenesis selections) was
pre-incubated for 30 minutes at room temperature in the dark with
40 nM of streptavidin XL.sup.ent!.TM. (CIS Bio International
611SAXLB).
[0498] All other conditions were as described for CAN022D10 epitope
competition assay. Data were analysed by calculating % deltaF and %
specific binding as previously described.
Inhibition of Endogenous IL-6 Induced VEGF Release from Human
Synovial Fibroblasts by Purified IgG
[0499] Samples of rheumatoid arthritis knees from total joint
replacement surgery were obtained in DMEM containing antibiotics.
Synovium bathed in media was dissected from the joint & finely
chopped. The synovial tissue was washed with media supplemented
with 10% FCS. The cell suspension was incubated in a collagenase
solution for 2 hours in a CO.sub.2 incubator at 37.degree. C. The
digested synovial cell suspension was disrupted by repeatedly
aspirating through a 10 ml pipette, cell strained & centrifuged
at 400 g at room temperature for 5 minutes. The cells were
resuspended in DMEM containing 10% FCS, passed through a cell
strainer, adjusted to 1.times.10.sup.6 cells per ml & incubated
in a CO.sub.2 incubator at 37.degree. C. in 225-cm.sup.2 cell
culture flasks (3001, CoStar Corning Inc.). Following adherence,
the majority of the medium was discarded, replaced with fresh &
returned to the incubator for long-term incubation. The cells were
examined on a weekly-basis & were passaged at confluence by
trypsinisation at a passage rate of 1 in 3.
[0500] Fibroblasts (P3-5) at confluence were removed from flasks by
incubating with 10 mL 0.1% trypsin-EDTA solution (25300-054, Gibco
Life Sciences) per flask for 5 to 10 minutes at 37.degree. C. An
equal volume of DMEM-based culture medium supplemented with 10% FCS
was added to the cells, which were then pelleted by centrifugation
at 330 g for 5 minutes at RT. After one wash step with DMEM-based
culture medium supplemented with 10% FCS, the cell suspension
(1.times.10.sup.5 cells per mL) was added (150 .mu.L per well) to
wells of sterile 96 well cell culture cluster flat bottom
polystyrene plates (3598, Corning CoStar) at 1.5.times.10.sup.4
cells per well. A further addition of DMEM-based culture media
supplemented with 10% FCS was added to each well (100 .mu.L per
well) to give a total volume of 250 .mu.L per well. The cells were
incubated at 37.degree. C. overnight to allow for adherence and
quiescence.
[0501] The 96-well plates were inspected to ensure that the cells
were confluent and in good condition (e.g. contamination-free).
Medium was then aspirated from the wells and 100 .mu.L of
DMEM-based culture medium supplemented with 10% FCS was immediately
added. To this, 50 .mu.L of DMEM-based culture medium supplemented
with 10% FCS containing either sample IgG or medium alone was added
to the wells (diluted 1 in 5 into assay).
[0502] This was followed by adding 50 .mu.L per well of DMEM-based
culture medium supplemented with 10% FCS containing recombinant
human soluble (rhs)IL-6R.alpha. (500 ng per mL; 12 nM) and
rhIL-1.beta. (50 pg per mL; 2.95 pM, diluted 1 in 5 into
assay).
[0503] In separate wells, 50 .mu.L of DMEM-based culture medium
supplemented with 10% FCS containing either; rh-IL-6 (0, 100 ng per
mL; 21.5 nM), sIL-6R.alpha. (500 ng per mL; 12 nM), rhIL-1.beta.
(50 pg per mL; 2.95 pM), or medium alone was added (diluted 1 in 5
into assay). Final volume in each well was 250 .mu.L.
[0504] The plates were incubated for 48 hours at 37.degree. C.
Incubations were performed in duplicate or triplicate wells as
described in the plate format. The plates were centrifuged at 330 g
for 5 minutes at RT and supernatant media was removed and stored at
-40.degree. C. in microtitre flat bottom plates (611F96,
Sterilin).
[0505] VEGF was measured using an ELISA (DY293B, R&D Systems)
following the manufacturers instructions. Briefly, ELISA plates
were coated with a mouse anti-human VEGF antibody overnight at
4.degree. C. and blocked with 1% BSA/PBS. Plates were washed with
0.05% Tween 20/PBS and incubated with culture supernatants of human
synovial derived fibroblasts and a biotinylated goat anti-human
VEGF antibody over night at room temperature. After washing, VEGF
was detected by using Streptavidin horseradish peroxidase. Plates
were developed using 1:1 H.sub.2O.sub.2:tetramethylbenzidine. The
reaction was stopped with 2 M H.sub.2SO.sub.4, and optical
densities were determined at 450 nm with the correction wavelength
set at 540 nm.
BIAcore Measurements
[0506] BIAcore studies were undertaken using a BIAcore 200.TM..
Antibodies were coupled to the surface of a CM-5 sensorchip using
an amine coupling kit to provide a surface density of 220-225 Ru.
Human IL-6 at a range of concentrations between 200 nM and 0.2 nM
in HBS-EP buffer were passed over the sensor chip surface. The
resulting sensorgrams were evaluated using BIA evaluation 3.1
software to calculate the k.sub.on, k.sub.off and K.sub.D values
for the antibodies tested.
IL-6 Mediated B9 Cell Proliferation Assay
[0507] B9 cells are a sub-clone of the murine B-cell hybridoma cell
line, B13.29, selected on the basis of their specific response to
IL-6.
[0508] B9 cells require IL-6 for survival and proliferation and
respond to very low concentrations of IL-6.
[0509] IL-6 induced B9 cell proliferation was assessed in the
presence of Antibody 18 and an isotype control (CAT-002). The
effects of a range of concentrations of each antibody
(1.times.10.sup.-13 M to 1.times.10.sup.-9 M) were assessed on an
IL-6 standard curve (concentration range 1.times.10.sup.-14 M to
1.times.10.sup.-9 M) Data points were in duplicate. B9
proliferation was determined after 4 days incubation by reduction
of alamar blue (fluorescence method).
[0510] B9 cells were cultured in RPMI-1640 containing 5% FCS, 2 mM
L-Glutamine and 50 .mu.M 2-mercaptoethanol. Cells were split every
2 to 4 days to a density of between 0.05.times.10.sup.6 mL.sup.-1
and 0.1.times.10.sup.6 mL.sup.-1 and supplemented with 5.times.10 M
human IL-6. Cells used for experiments were not supplemented with
IL-6 for at least 48 hours prior to experiment but had been
supplemented within 96 hours of experiment. Cells used in the assay
were taken from a stock flask with a density of no greater than
0.8.times.10.sup.6 mL.sup.-1.
[0511] Each antibody was diluted from stock solutions to 10.times.
the maximum required assay concentration by appropriate dilutions
in assay media (RPMI+5% FCS, 2 mM L-Glutamine, 50 .mu.M
2-mercaptoethanol, penicillin 100 UmL.sup.-1 and streptomycin 100
mgmL.sup.-1). Further 10 fold dilutions in culture media were
carried out to obtain the required concentrations of each
antibody.
[0512] IL-6 was reconstituted from a lyophilised powder to a
1.times.10.sup.-5 M solution by addition of an appropriate volume
of sterile PBS+0.1% BSA. A further dilution to 1.times.10.sup.-8 M
was carried out in culture media. 1.times.10.sup.-8 M aliquots were
stored frozen until required. On the day of assay 1.times.10.sup.-8
M aliquots were diluted as necessary to achieve the range of
solutions at 10.times. final assay concentration required.
[0513] The required volume of cells was removed from culture flasks
and centrifuged at 300 g for 8 minutes. Supernatants were removed
and the cells re-suspended in an appropriate volume of culture
media to achieve a cell density of 0.5.times.10.sup.6
mL.sup.-1.
[0514] Assays were performed in flat-bottomed, tissue culture
treated, polystyrene 96 well plates. The final assay volume was 200
.mu.L. 20 .mu.L of 10.times. antibody (Antibody 18 or CAT-002)
solution or culture media was added to the appropriate wells of
each plate followed by a further 140 .mu.L of culture media and 20
.mu.L of the appropriate concentration of IL-6 or culture
media.
[0515] Plates were placed in a humidified 5% CO.sub.2, 37.degree.
C. incubator for 2 hours. 20 .mu.L of cells was then added to each
well. Final number of cells per well was 10000. Plates were then
returned to the incubator for 4 days. Cell proliferation was
assessed by incorporation of alamar blue. 10% v/v alamar blue was
added to each well and the plates returned to the incubator for 6
hours. Plates were then read on a spectrofluorimeter measuring
fluorescence at 590 nm following excitation at 544 nm Raw data were
normalised to the control IL-6 curve on the each plate such that
maximum fluorescence was defined as 100% and the basal fluorescence
0%. Normalised data was fitted using the non linear regression,
sigmoidal dose-response (variable slope) fitting programme in Graph
Pad Prism 4.01. Control pEC.sub.50 values and pEC.sub.50 values in
the presence of each concentration of antibody were used to
determine dose ratios (DR). Kb values were determined for the
lowest concentration of antibody which elicited a 3-fold or greater
shift in the IL-6 concentration-effect curve using the chemical
antagonism equation below:
K.sub.b=([Ab]/(DR-1))
[0516] (Kenakin T P, In: Pharmacologic Analysis of Drug-Receptor
Interactions. 1st ed. New York: Raven Press; 1987. p. 205-24.)
IL-6 Mediated SKW6.4 Cell IgM Release Assay
[0517] IL-6 is involved in the final maturation of B cells into
antibody producing cells (B-lymphocyte differentiation). SKW cells
have been used previously for the study of B cell responses (Nawata
et al., Ann. N.Y. Acad. Sci. 557:230-238. 1989). Auto-antibody
production in rheumatoid arthritis is mostly of the IgM class.
SKW6.4 is a clonal IgM secreting human lymphoblastoid B cell line.
Cells were sourced from ATCC, reference #TIB 215. Upon stimulation
with IL-6 these cells secrete IgM, thus this assay was perceived to
be relevant to rheumatoid arthritis.
[0518] IL-6 induced SKW6.4 cell IgM secretion was assessed in the
presence of CAT6001 and CAT-002 (isotype control). The effects of a
range of concentrations of each antibody (1.times.10.sup.-12.5 M to
1.times.10.sup.-8 M) were assessed in the presence of 100 pM IL-6.
Data points were in duplicate. IgM secretion in the cell
supernatants was determined after 4 days incubation using
anti-human IgM ELISA assay.
[0519] SKW 6.4 cells were cultured in RPMI1640 containing 2 mM
L-Glutamine and 10% (v/v) foetal calf serum at 37.degree. C. at
95/5% (v/v) air/CO.sub.2 in 95% relative humidity. The cells were
maintained between 0.4 and 2.times.10.sup.6 cells/ml. For routine
cell passage, cells were harvested by centrifugation at 300.times.g
for 5 minutes at room temperature, spent medium was removed and the
cells re-suspended in the required volume of fresh media.
[0520] Each antibody was diluted from stock solutions to 50.times.
the maximum required assay concentration by appropriate dilutions
in assay media (RPMI+10% FCS, 2 mM L-Glutamine). Further 10 fold
dilutions in culture media were carried out to obtain the required
concentrations of each antibody.
[0521] Assays were performed in flat-bottomed, tissue culture
treated, polystyrene 96 well plates. SKW 6.4 cell stocks were
diluted to a cell density of 0.3.times.10.sup.6 ml.sup.-1 in fresh
media, and plated at 100 .mu.l/well, (30,000 cells per well). 2
.mu.l of antibody, at the indicated final concentration, followed
by 2 .mu.l of IL-6 at a final concentration of 100 pM was then
added to each well.
[0522] Plates were then returned to the incubator at 37.degree. C.
5% CO.sub.2. Cell-free supernatants were harvested after 4 days
incubation by centrifugation and then either assayed by IgM ELISA
on the day of harvest or frozen at -20.degree. C. prior to further
analysis.
[0523] An ELISA was generated using a pair of antibodies from
Serotec. The coating antibody was Mouse anti-human IgM (MCA1662)
and the detection antibody was Goat anti-human IgM: HRP linked
(STAR98P). The assay was optimised by standard methods to give a
good signal to noise ratio using coating antibody @ 1:2000 dilution
(5 .mu.g/ml) and detection antibody @ 1:3500 dilution (200
ng/ml).
[0524] IgM standard solution (Cat #PHP003 Human M Kappa purified
protein) was purchased from Serotec to generate a standard
curve.
[0525] Data was analysed using a polynomial fit for the IgM
standard curve data using a standard fitting programme. The
percentage inhibition of each antibody sample against the control
IgM production in the absence of antibody was calculated and
IC.sub.50 values were generated.
Generation of IL-6 and IL-6 Mutant Proteins for Epitope Mapping
[0526] Cloning of Human and Cyno IL-6 cDNA
[0527] The sequences of human and macaque IL-6 were obtained from
Embl (Accession No: BC015511 and AB000554 for human and cyno,
respectively). Using these sequences oligonucleotide primers were
designed to amplify the cDNA encoding human & macaque IL-6. The
N-terminal primers were hIL6-5'NdeI and macIL65' NdeI for human and
cyno respectively and macIL63'NheI was used as the C terminal
primer for both (See Table 11 for oligonucleotides sequences).
TABLE-US-00011 TABLE 11 Primer sequences Primer Sequence
macIL6_5'NdeI 5' TTATCAT-ATGGTACTCCCAGGAGAAGA TTCCAA 3' (SEQ ID NO:
183) macIL6_3'NheI 5' TTATGCTAGC-CTACATTTGCCGAAGAG CCC 3' (SEQ ID
NO: 184) hIL6_5'NdeI 5' TTATACATATG-GTACCCCCAGGAGAAG ATTCC 3' (SEQ
ID NO: 185)
[0528] PCVR reactions to amplify the two cDNAs were carried out.
The template for each PCR reaction was 10 ng of cDNA obtained from
human Liver and cynomolgus liver respectively. The amplified cDNA
from each reaction was purified and cloned into pCR4blunt topo
(Invitrogen) using the topoisomerase ligation reaction according to
the manufacturer.
[0529] Positive clones were identified and sequenced. The resulting
cDNAs 10 were sub-cloned using standard techniques into various E.
coli T7-promoter expression vectors in such a way that the cDNA
encoding mature human or cynomolgus IL-6 were fused at the
N-terminus with either an N-terminal HIS6-FLAG tag immediately
upstream of the N-terminal valine of mature IL-6.
Generating Mutants
[0530] Site directed mutagenesis was performed using a Quikchange
XL kit from Stratagene according to the manufacturer's protocol.
Mutagenesis primer design was performed according to the
manufacturer's protocol. Mutagenesis reactions were carried out
according to the protocol using plasmid pT7flagHISIL-6 as template.
This was followed by subsequent DpnI digestion and transformation
into chemically competent Top10 cells with selection on agar plates
containing appropriate antibiotics at 37.degree. C. overnight. For
each individual mutagenesis reaction several clones were sequenced
and plasmid DNA of one correct clone from each reaction was
retained for further use.
Expression of IL-6 and IL-6 Mutant Proteins
[0531] The IL-6 expression plasmids were transformed into
chemically competent BL21 (DE3) star cells (Invitrogen) using the
manufacturer's method. Transformed cells were used to inoculate 1 L
cultures of Terrific Broth and these were incubated on an orbital
incubator at 37.degree. C., until the A600 reached 0.5. IPTG was
then added to 0.25 mM and incubation continued overnight at
22.degree. C. The cells were harvested by centrifugation and the
cell pellets were stored at -80.degree. C.
Purification of IL-6 and IL-6 Mutant Proteins
[0532] The cell pellets were thawed and resuspended in 50 ml per
pellet of 50 mM potassium phosphate, pH7.4, 10 mM imidazole, 0.3 M
NaCl, 5 mM beta-mercaptoethanol, 10% glycerol (buffer A)+Complete
EDTA-free protease inhibitors (Roche). The cells were lysed by
sonication for 3.times.30 seconds on ice. The lysate was
centrifuged at 100,000 g and 4.degree. C. for 30 minutes and the
supernatant was subjected to Ni NTA affinity chromatography. A 5 ml
column of Ni-NTA Superflow (Qiagen) was equilibrated at 3 ml/min
with (buffer A). The IL-6 sample was loaded and the column was
washed with 10 column volumes of 15 mM imidazole in buffer A. This
was followed by a 10 column volume wash with 30 mM imidazole in
buffer A. IL-6 was eluted from the column using a 5 column volume
wash in the upward flow direction with 0.3 M imidazole in buffer A.
10 ml fractions were collected during the wash steps and 5 ml
fractions were collected during the elution step. The column was
run at 4.degree. C. using the AKTA Explorer100 Air. Fractions
containing the purified IL-6 protein were pooled and dialysed
overnight at 4.degree. C. against 5 L of PBS.
[0533] The dialysed IL-6 proteins were further purified using gel
filtration chromatography. For each purification the dialysed IL-6
protein was centrifuged at 100,000 g and 4.degree. C. for 20
minutes. Up to 13 ml was applied to a 318 ml Superdex 20026/60
column (GE Healthcare) that had been equilibrated in PBS at 2.5
ml/min. The column was run at 4.degree. C. using an AKTA Purifier.
Fractions containing the monomeric IL-6 protein peak were pooled
for further analysis.
[0534] Each protein was checked for purity using standard
SDS-chromatography, the protein concentration was measured and
Q-ToF mass spectroscopy was used to measure the mass of the
protein. Purified IL-6 was frozen in liquid nitrogen and stored at
-80.degree. C.
Materials and Methods for In Vivo Studies
[0535] Animals were randomly assigned to into test groups. The mice
in each test group were then treated daily with set sub-cutaneous
doses (10 ml/kg) of either vehicle control (0.05% BSA in PBS) or
467 m/kg IgG1 isotype control or antibody 18 (range from 467 m/kg
to 8 .mu.g/kg). At the same time the mice were given an
intra-peritoneal injection (10 ml/kg) b.i.d. of either vehicle
control (0.05% BSA in PBS) or 12 .mu.g/kg human recombinant
IL-6.
[0536] On day 7, two hours following the final IL-6 dose at 09:00
h, the mice were sacrificed and terminal blood samples were taken.
The blood was transferred to Lab Tek 1 ml EDTA blood tubes, which
were placed on a roller for 5 minutes. Samples were then kept on
ice until used. Differential cell counts were performed using a
Sysmex cell counter. The remainder of the sample was transferred to
an eppendorf tube and spun (300 g, 5 mins) to obtain plasma which
was sub aliquoted and stored at -20.degree. C. until anlaysed for
Haptoglobin levels.
[0537] The haptoglobin assay was carried out as per instructions
provided in the PHASE.TM. RANGE TriDelta Format kit by Biognosis
(Hailsham, UK; cat. no. TP-801).
[0538] All results were expressed as mean.+-.SEM. Data analysis was
by unpaired T-test or one-way ANOVA followed by Dunnett's test
(GraphPad Instat).
Sequences
[0539] VH domain, VL domain and CDR sequences of binding members
are shown in the appended sequence listing, in which SEQ ID NOS
correspond as follows:
TABLE-US-00012 1 CAN022D10 VH nucleotide 2 CAN022D10 VH amino acid
3 CAN022D10 VH CDR 1 aa 4 CAN022D10 VH CDR 2 aa 5 CAN022D10 VH CDR
3 aa 6 CAN022D10 VL nucleotide 7 CAN022D10 VL amino acid 8
CAN022D10 VL CDR 1 aa 9 CAN022D10 VL CDR 2 aa 10 CAN022D10 VL CDR 3
aa 11 Antibody 2 VH nucleotide 12 Ab 2 VH amino acid 13 Ab 2 VH CDR
1 amino acid 14 Ab 2 VH CDR 2 amino acid 15 Ab 2 VH CDR 3 amino
acid 16 Ab 2 VL nucleotide 17 Ab 2 VL amino acid 18 Ab 2 VL CDR 1
amino acid 19 Ab 2 VL CDR 2 amino acid 20 Ab 2 VL CDR 3 amino acid
21 Antibody 3 VH nucleotide 22 Ab 3 VH amino acid 23 Ab 3 VH CDR 1
amino acid 24 Ab 3 VH CDR 2 amino acid 25 Ab 3 VH CDR 3 amino acid
26 Ab 3 VL nucleotide 27 Ab 3 VL amino acid 28 Ab 3 VL CDR 1 amino
acid 29 Ab 3 VL CDR 2 amino acid 30 Ab 3 VL CDR 3 amino acid 31
Antibody 4 VH nucleotide 32 Ab 4 VH amino acid 33 Ab 4 VH CDR 1
amino acid 34 Ab 4 VH CDR 2 amino acid 35 Ab 4 VH CDR 3 amino acid
36 Ab 4 VL nucleotide 37 Ab 4 VL amino acid 38 Ab 4 VL CDR 1 amino
acid 39 Ab 4 VL CDR 2 amino acid 40 Ab 4 VL CDR 3 amino acid 41
Antibody 5 VH nucleotide 42 Ab 5 VH amino acid 43 Ab 5 VH CDR 1
amino acid 44 Ab 5 VH CDR 2 amino acid 45 Ab 5 VH CDR 3 amino acid
46 Ab 5 VL nucleotide 47 Ab 5 VL amino acid 48 Ab 5 VL CDR 1 amino
acid 49 Ab 5 VL CDR 2 amino acid 50 Ab 5 VL CDR 3 amino acid 51
Antibody 7 VH nucleotide 52 Ab 7 VH amino acid 53 Ab 7 VH CDR 1
amino acid 54 Ab 7 VH CDR 2 amino acid 55 Ab 7 VH CDR 3 amino acid
56 Ab 7 VL nucleotide 57 Ab 7 VL amino acid 58 Ab 7 VL CDR 1 amino
acid 59 Ab 7 VL CDR 2 amino acid 60 Ab 7 VL CDR 3 amino acid 61
Antibody 8 VH nucleotide 62 Ab 8 VH amino acid 63 Ab 8 VH CDR 1
amino acid 64 Ab 8 VH CDR 2 amino acid 65 Ab 8 VH CDR 3 amino acid
66 Ab 8 VL nucleotide 67 Ab 8 VL amino acid 68 Ab 8 VL CDR 1 amino
acid 69 Ab 8 VL CDR 2 amino acid 70 Ab 8 VL CDR 3 amino acid 71
Antibody 10 VH nucleotide 72 Ab 10 VH amino acid 73 Ab 10 VH CDR 1
amino acid 74 Ab 10 VH CDR 2 amino acid 75 Ab 10 VH CDR 3 amino
acid 76 Ab 10 VL nucleotide 77 Ab 10 VL amino acid 78 Ab 10 VL CDR
1 amino acid 79 Ab 10 VL CDR 2 amino acid 80 Ab 10 VL CDR 3 amino
acid 81 Antibody 14 VH nucleotide 82 Ab 14 VH amino acid 83 Ab 14
VH CDR 1 amino acid 84 Ab 14 VH CDR 2 amino acid 85 Ab 14 VH CDR 3
amino acid 86 Ab 14 VL nucleotide 87 Ab 14 VL amino acid 88 Ab 14
VL CDR 1 amino acid 89 Ab 14 VL CDR 2 amino acid 90 Ab 14 VL CDR 3
amino acid 91 Antibody 16 VH nucleotide 92 Ab 16 VH amino acid 93
Ab 16 VH CDR 1 amino acid 94 Ab 16 VH CDR 2 amino acid 95 Ab 16 VH
CDR 3 amino acid 96 Ab 16 VL nucleotide 97 Ab 16 VL amino acid 98
Ab 16 VL CDR 1 amino acid 99 Ab 16 VL CDR 2 amino acid 100 Ab 16 VL
CDR 3 amino acid 101 Antibody 17 VH nucleotide 102 Ab 17 VH amino
acid 103 Ab 17 VH CDR 1 amino acid 104 Ab 17 VH CDR 2 amino acid
105 Ab 17 VH CDR 3 amino acid 106 Ab 17 VL nucleotide 107 Ab 17 VL
amino acid 108 Ab 17 VL CDR 1 amino acid 109 Ab 17 VL CDR 2 amino
acid 110 Ab 17 VL CDR 3 amino acid 111 Antibody 18 VH nucleotide
112 Ab 18 VH amino acid 113 Ab 18 VH CDR 1 amino acid 114 Ab 18 VH
CDR 2 amino acid 115 Ab 18 VH CDR 3 amino acid 116 Ab 18 VL
nucleotide 117 Ab 18 VL amino acid 118 Ab 18 VL CDR 1 amino acid
119 Ab 18 VL CDR 2 amino acid 120 Ab 18 VL CDR 3 amino acid 121
Antibody 19 VH nucleotide 122 Ab 19 VH amino acid 123 Ab 19 VH CDR
1 amino acid 124 Ab 19 VH CDR 2 amino acid 125 Ab 19 VH CDR 3 amino
acid 126 Ab 19 VL nucleotide 127 Ab 19 VL amino acid 128 Ab 19 VL
CDR 1 amino acid 129 Ab 19 VL CDR 2 amino acid 130 Ab 19 VL CDR 3
amino acid 131 Antibody 21 VH nucleotide 132 Ab 21 VH amino acid
133 Ab 21 VH CDR 1 amino acid 134 Ab 21 VH CDR 2 amino acid 135 Ab
21 VH CDR 3 amino acid 136 Ab 21 VL nucleotide 137 Ab 21 VL amino
acid 138 Ab 21 VL CDR 1 amino acid 139 Ab 21 VL CDR 2 amino acid
140 Ab 21 VL CDR 3 amino acid 141 Antibody 22 VH nucleotide 142 Ab
22 VH amino acid 143 Ab 22 VH CDR 1 amino acid 144 Ab 22 VH CDR 2
amino acid 145 Ab 22 VH CDR 3 amino acid 146 Ab 22 VL nucleotide
147 Ab 22 VL amino acid 148 Ab 22 VL CDR 1 amino acid 149 Ab 22 VL
CDR 2 amino acid 150 Ab 22 VL CDR 3 amino acid 151 Antibody 23 VH
nucleotide 152 Ab 23 VH amino acid 153 Ab 23 VH CDR 1 amino acid
154 Ab 23 VH CDR 2 amino acid 155 Ab 23 VH CDR 3 amino acid 156 Ab
23 VL nucleotide 157 Ab 23 VL amino acid 158 Ab 23 VL CDR 1 amino
acid 159 Ab 23 VL CDR 2 amino acid 160 Ab 23 VL CDR 3 amino acid
161 Full length human IL-6 amino acid 162 HIS FLAG tagged human
IL-6 163 Soluble IL-6Ra (human) 164 Transmembrane IL-6Ra (human)
165 Mature human IL-6 amino acid 166 Human gp130 167 Germlined VH
FR1 168 Germlined VH FR2 169 Germlined VH FR3 170 Germlined VH FR4
171 Germlined VL FR1 172 Germlined VL FR1 173 Germlined VL FR1 174
Germlined VL FR1 175 F102E mutant IL-6 176 S204E mutant IL-6 177
R207E mutant IL-6 178 F106E mutant IL-6 179 Q211A mutant IL-6 180
R58E mutant IL-6 181 E200W mutant IL-6 182 R207L mutant IL-6 183
primer macIL6_5'NdeI 184 primer macIL6_3'NheI 185 primer
hIL6_5'NdeI
Sequences of antibodies 7, 10, 17 and 18 are germlined.
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TABLE-US-00013 [0674] TABLE 7 HCDR1 HCDR2 HCDR3 Kabat numbering 31
32 33 34 35 50 51 52 52A 53 54 55 56 57 58 59 60 61 62 63 64 65 95
96 97 CAN022D10 S N Y M I D L Y Y Y A G D T Y Y A D S V K G W A D
Antibody 2 T R Antibody 3 V Antibody 4 T Antibody 5 Antibody 7
Antibody 8 Antibody 10 E E Antibody 14 Antibody 16 Antibody 17
Antibody 18 Antibody 19 Antibody 21 Antibody 22 Antibody 23 HCDR3
LCDR1 Kabat numbering 98 99 100 100A 100B 100C 100D 101 102 24 25
26 27 28 29 30 31 32 33 34 CAN022D10 D H Y Y Y I -- D V R A S Q G I
S S W L A Antibody 2 G T Antibody 3 H Antibody 4 G A Antibody 5 G A
Antibody 7 P A W V L Antibody 8 P R H Antibody 10 E G R G Antibody
14 N P H I Antibody 16 P P L Antibody 17 P P M Antibody 18 P P W L
Antibody 19 P S H L I Antibody 21 P S H Antibody 22 N N T Y I
Antibody 23 A P W V L LCDR2 LCDR3 Kabat numbering 50 51 52 53 54 55
56 89 90 91 92 93 94 95 96 97 CAN022D10 K A S T L E S Q Q S Y S T P
W T Antibody 2 A Antibody 3 Antibody 4 A Antibody 5 A Antibody 7 W
L G -- G S Antibody 8 W L G -- G S Antibody 10 W L G -- G S
Antibody 14 A A H A A Antibody 16 W L G -- G S Antibody 17 W L G --
G S Antibody 18 W L G -- G S Antibody 19 W L G -- G S Antibody 21 W
L G -- G S Antibody 22 A A H A A Antibody 23 W L G -- G S
Sequence CWU 1
1
1851360DNAHomo sapiensCAN022D10 1gaagtgcagc tggtgcagtc tgggggaggc
ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccatcagc
agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg ggctggagtg
ggtctccgat ctttattatt atgctggtga cacatattac 180gcagactccg
tgaagggccg attcaccatg tccagagaca tttccaagaa caccgtgtat
240cttcaaatga acagcctgag agccgaggac acgggtgtct attattgtgc
gagatgggcc 300gatgaccact actattacat tgacgtctgg ggcaggggca
ccctggtcac cgtctcgagt 3602120PRTHomo sapiensCAN022D10 2Glu Val Gln
Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25 30Tyr
Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr Val
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Val
Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Tyr Tyr Tyr Ile Asp
Val Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12035PRTHomo sapiensCAN022D10 3Ser Asn Tyr Met Ile1 5417PRTHomo
sapiensCAN022D10 4Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala
Asp Ser Val Lys1 5 10 15Gly511PRTHomo sapiensCAN022D10 5Trp Ala Asp
Asp His Tyr Tyr Tyr Ile Asp Val1 5 106324DNAHomo sapiensCAN022D10
6gacatcgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggccagtca gggtattagt agctggttgg cctggtatca gcagaaacca
120gggagagccc ctaaggtctt gatctataag gcatctactt tagaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag
agttacagta ccccgtggac gttcggccaa 300gggaccaagc tggagatcaa acgt
3247108PRTHomo sapiensCAN022D10 7Asp Ile Val Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp 85 90 95Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105811PRTHomo
sapiensCAN022D10 8Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5
1097PRTHomo sapiensCAN022D10 9Lys Ala Ser Thr Leu Glu Ser1
5109PRTHomo sapiensCAN022D10 10Gln Gln Ser Tyr Ser Thr Pro Trp Thr1
511360DNAHomo sapiensAntibody 02 11gaggtgcagc tggtgcagtc agggggaggc
ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccatcagc
agcaactaca tgacttgggt ccgtcaggct 120ccagggaagg ggctggagtg
ggtctccgat ctttattatt atgctggtga cacatattac 180gcagactccg
tgaggggccg attcaccatg tccagagaca tttccaagaa caccgtgtat
240cttcaaatgg acagcctgag agccgaggac acgggtgtct attattgtgc
gagatgggcc 300gatggccact actattacat tgacgtctgg ggcgggggca
ccctggtcac cgtctcgagt 36012120PRTHomo sapiensAntibody 02 12Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25
30Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Arg Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Gly
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Gly His Tyr Tyr Tyr Ile
Asp Val Trp Gly Gly 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120135PRTHomo sapiensAntibody 02 13Ser Asn Tyr Met Thr1
51417PRTHomo sapiensAntibody 02 14Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Arg1 5 10 15Gly1511PRTHomo
sapiensAntibody 02 15Trp Ala Asp Gly His Tyr Tyr Tyr Ile Asp Val1 5
1016324DNAHomo sapiensAntibody 02 16gacatcgtga tgacccagtc
cccttccacc ctgtctgcat ctgtaggaga cagagtcact 60atcacttgcc gggccagtca
gggtattagt agctggttga cctggtatca gcagaaacca 120gggagagccc
ctaaggtctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcaccctca ccatcagcag
tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagtg
ccccgtggac gttcggccaa 300gggaccaagc tggagctcaa acgt 32417108PRTHomo
sapiensAntibody 02 17Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Thr Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Leu Lys Arg 100 1051811PRTHomo sapiensAntibody
02 18Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Thr1 5 10197PRTHomo
sapiensAntibody 02 19Lys Ala Ser Thr Leu Glu Ser1 5209PRTHomo
sapiensAntibody 02 20Gln Gln Ser Tyr Ser Ala Pro Trp Thr1
521360DNAHomo sapiensAntibody 03 21caggtacagc tggtgcagtc tgggggaggc
ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccatcagc
agcaactaca tggtttgggt ccgtcaggct 120ccggggaagg ggctggagtg
ggtctccgat ctttattatt atgctggtga cacatattac 180gcagactccg
tgaagggccg attcaccgtg tccagagaca tttccaagaa caccgtgtat
240cttcaaatga acagcctgag agccgaggac acgggtgtct attattgtgc
gagatgggcc 300gatgaccact actatcacat tgacgtctgg ggcaggggca
ccctggtcac cgtctcgagt 36022120PRTHomo sapiensAntibody 03 22Gln Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25
30Tyr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Ile Ser Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Tyr Tyr His Ile
Asp Val Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120235PRTHomo sapiensAntibody 03 23Ser Asn Tyr Met Val1
52417PRTHomo sapiensAntibody 03 24Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly2511PRTHomo
sapiensAntibody 03 25Trp Ala Asp Asp His Tyr Tyr His Ile Asp Val1 5
1026324DNAHomo sapiensAntibody 03 26gacatcgtga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggtattagt agctggttgg cctggtatca gcagaaacca 120gggagagccc
ctaaggcctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagta
ccccgtggac gttcggccaa 300gggaccaagc tggagatcaa acgt 32427108PRTHomo
sapiensAntibody 03 27Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Ala Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg 100 1052811PRTHomo sapiensAntibody
03 28Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 10297PRTHomo
sapiensAntibody 03 29Lys Ala Ser Thr Leu Glu Ser1 5309PRTHomo
sapiensAntibody 03 30Gln Gln Ser Tyr Ser Thr Pro Trp Thr1
531360DNAHomo sapiensAntibody 04 31gaggtgcagc tggtgcagtc agggggaggc
ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccgtcagc
agcaactaca tgacttgggt ccgacaggct 120ccagggaagg ggctggagtg
ggtctccgat ctttattatt atgctggcga cacgtattac 180gcagactccg
tgaagggccg attcaccatg tccagagaca tttccaagaa caccgtgtac
240cttcaaatga acagcctaag agccgaggac acgggtgtct attattgtgc
gagatgggcc 300gatggccact actattacgc tgacgtctgg ggcaggggca
ccctggtctc cgtctcgagt 36032120PRTHomo sapiensAntibody 04 32Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25
30Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Gly His Tyr Tyr Tyr Ala
Asp Val Trp Gly Arg 100 105 110Gly Thr Leu Val Ser Val Ser Ser 115
120335PRTHomo sapiensAntibody 04 33Ser Asn Tyr Met Thr1
53417PRTHomo sapiensAntibody 04 34Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly3511PRTHomo
sapiensAntibody 04 35Trp Ala Asp Gly His Tyr Tyr Tyr Ala Asp Val1 5
1036324DNAHomo sapiensAntibody 04 36gacatcgtga tgacccagtc
tccctccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggaattagt agctggttgg cctggtatca gcagaaacca 120gggagagccc
ctaaggtctt gatctataag gcatctacgt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagatttcg catcttacta ctgtcaacag agttacagtg
ccccgtggac gttcggccaa 300gggaccaagc tggagctcaa acgt 32437108PRTHomo
sapiensAntibody 04 37Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Ser
Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Leu Lys Arg 100 1053811PRTHomo sapiensAntibody
04 38Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 10397PRTHomo
sapiensAntibody 04 39Lys Ala Ser Thr Leu Glu Ser1 5409PRTHomo
sapiensAntibody 04 40Gln Gln Ser Tyr Ser Ala Pro Trp Thr1
541360DNAHomo sapiensAntibody 05 41gaggtgcagc tggtgcagtc tgggggaggc
ttgatccagc cgggggggtc cctgagactc 60tcctgtgcgg cctctgggtt caccatcagc
agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg ggctggagtg
ggtctccgat ctttattatt atgctggcga cacgtattac 180gcagactccg
tgaagggccg attcaccatg tccagagaca tttccaagaa caccgtgtac
240cttcaaatga acagcctgag agccgaggac acgggtgtct attattgtgc
gagatgggcc 300gatggccact actattacgc tgacgtctgg ggcaggggca
ccctggtctc cgtctcgagt 36042120PRTHomo sapiensAntibody 05 42Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25
30Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Gly His Tyr Tyr Tyr Ala
Asp Val Trp Gly Arg 100 105 110Gly Thr Leu Val Ser Val Ser Ser 115
120435PRTHomo sapiensAntibody 05 43Ser Asn Tyr Met Ile1
54417PRTHomo sapiensAntibody 05 44Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly4511PRTHomo
sapiensAntibody 05 45Trp Ala Asp Gly His Tyr Tyr Tyr Ala Asp Val1 5
1046324DNAHomo sapiensAntibody 05 46gacatcgtga tgacccagtc
tccccccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggaattagt agctggttgg cctggtatca gcagaaacca 120gggagagccc
ctaaggtctt gatctataag gcatctacat tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagatttcg catcttacta ctgtcaacag agttacagtg
ccccgtggac gtttggccaa 300gggaccaagc tggagatcaa acgt 32447108PRTHomo
sapiensAntibody 05 47Asp Ile Val Met Thr Gln Ser Pro Pro Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Ser
Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg 100 1054811PRTHomo sapiensAntibody
05 48Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 10497PRTHomo
sapiensAntibody 05 49Lys Ala Ser Thr Leu Glu Ser1 5509PRTHomo
sapiensAntibody 05 50Gln Gln Ser Tyr Ser Ala Pro Trp Thr1
551360DNAHomo sapiensAntibody 07 51gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccatcagc
agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg ggctggagtg
ggtctccgat ctttattatt atgctggtga cacatattac 180gcagactccg
tgaagggccg attcaccatg tccagagaca tttccaagaa caccgtgtat
240cttcaaatga acagcctgag agccgaggac acggctgtct attattgtgc
gagatgggcc 300gatgaccacc cggcctgggt ggacctctgg ggcaggggca
ccctggtcac cgtctcctca 36052120PRTHomo sapiensAntibody 07 52Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25
30Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Pro Ala Trp Val
Asp Leu Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120535PRTHomo sapiensAntibody 07 53Ser Asn Tyr Met Ile1
55417PRTHomo sapiensAntibody 07 54Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly5511PRTHomo
sapiensAntibody 07 55Trp Ala Asp Asp His Pro Ala Trp Val Asp Leu1 5
1056321DNAHomo
sapiensAntibody 07 56gacatccaga tgacccagtc tccttccacc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gggtattagt agctggttgg
cctggtatca gcagaaacca 120gggaaagccc ctaaggtctt gatctataag
gcatctactt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagaa ttcactctca ccatcagcag tctgcaacct 240gatgattttg
caacttacta ctgtcaacag agttggctcg gcgggtcgtt cggccaaggg
300accaagctgg agatcaaacg t 32157107PRTHomo sapiensAntibody 07 57Asp
Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu
Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Trp Leu Gly Gly Ser 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 1055811PRTHomo sapiensAntibody 07 58Arg Ala Ser Gln Gly Ile
Ser Ser Trp Leu Ala1 5 10597PRTHomo sapiensAntibody 07 59Lys Ala
Ser Thr Leu Glu Ser1 5608PRTHomo sapiensAntibody 07 60Gln Gln Ser
Trp Leu Gly Gly Ser1 561360DNAHomo sapiensAntibody 08 61gaagtgcagc
tggtgcagtc tgggggaggc ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag
cctctgggtt caccatcagc agcaactaca tgatttgggt ccgtcaggct
120ccagggaagg ggctggagtg ggtctccgat ctttattatt atgctggtga
cacatattac 180gcagactccg tgaagggccg attcaccatg tccagagaca
tttccaagaa caccgtgtat 240cttcaaatga acagcctgag agccgaggac
acgggtgtct attattgtgc gagatgggcc 300gatgaccacc cccggtacat
cgaccactgg ggcaggggca ccctggtcac cgtctcgagt 36062120PRTHomo
sapiensAntibody 08 62Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu
Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Ile Ser Ser Asn 20 25 30Tyr Met Ile Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly
Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Met Ser
Arg Asp Ile Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala
Asp Asp His Pro Arg Tyr Ile Asp His Trp Gly Arg 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 120635PRTHomo sapiensAntibody 08 63Ser
Asn Tyr Met Ile1 56417PRTHomo sapiensAntibody 08 64Asp Leu Tyr Tyr
Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly6511PRTHomo sapiensAntibody 08 65Trp Ala Asp Asp His Pro Arg
Tyr Ile Asp His1 5 1066321DNAHomo sapiensAntibody 08 66gacatcgtga
tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggccagtca gggtattagt agctggttgg cctggtatca gcagaaacca
120gggagagccc ctaaggtctt gatctataag gcatctactt tagaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag
agttggctcg gcgggtcgtt cggccaaggg 300accaagctgg agatcaaacg t
32167107PRTHomo sapiensAntibody 08 67Asp Ile Val Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser
Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90
95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 1056811PRTHomo
sapiensAntibody 08 68Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5
10697PRTHomo sapiensAntibody 08 69Lys Ala Ser Thr Leu Glu Ser1
5708PRTHomo sapiensAntibody 08 70Gln Gln Ser Trp Leu Gly Gly Ser1
571360DNAHomo sapiensAntibody 10 71gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccatcagc
agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg ggctggagtg
ggtctccgat ctttattatt atgctggtga cacatattac 180gcagactccg
tgaagggccg attcaccatg tccagagaca tttccaagaa caccgtgtat
240cttcaaatga acagcctgag agccgaggac acggctgtct attattgtgc
gagatgggag 300gaggagggga gggggtacat tgacgtctgg ggcaggggca
ccctggtcac cgtctcctca 36072120PRTHomo sapiensAntibody 10 72Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25
30Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Glu Glu Glu Gly Arg Gly Tyr Ile
Asp Val Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120735PRTHomo sapiensAntibody 10 73Ser Asn Tyr Met Ile1
57417PRTHomo sapiensAntibody 10 74Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly7511PRTHomo
sapiensAntibody 10 75Trp Glu Glu Glu Gly Arg Gly Tyr Ile Asp Val1 5
1076321DNAHomo sapiensAntibody 10 76gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggtattagt agctggttgg cctggtatca gcagaaacca 120gggaaagccc
ctaaggtctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
tctgcaacct 240gatgattttg caacttacta ctgtcaacag agttggctcg
gcgggtcgtt cggccaaggg 300accaagctgg agatcaaacg t 32177107PRTHomo
sapiensAntibody 10 77Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90 95Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 1057811PRTHomo sapiensAntibody 10
78Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 10797PRTHomo
sapiensAntibody 10 79Lys Ala Ser Thr Leu Glu Ser1 5808PRTHomo
sapiensAntibody 10 80Gln Gln Ser Trp Leu Gly Gly Ser1 581363DNAHomo
sapiensAntibody 14 81gaagtgcagc tggtgcagtc tgggggaggc ttgatccagc
cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccatcagc agcaactaca
tgatttgggt ccgtcaggct 120ccagggaagg ggctggagtg ggtctccgat
ctttattatt atgctggtga cacatattac 180gcagactccg tgaagggccg
attcaccatg tccagagaca tttccaagaa caccgtgtat 240cttcaaatga
acagcctgag agccgaggac acgggtgtct attattgtgc gagatgggcc
300gatgaccaca actaccccca cattgacgtc tggggcaggg gcaccctggt
caccgtctcg 360agt 36382121PRTHomo sapiensAntibody 14 82Glu Val Gln
Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25 30Tyr
Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr Val
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Val
Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Asn Tyr Pro His Ile
Asp Val Trp Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser 115
120835PRTHomo sapiensAntibody 14 83Ser Asn Tyr Met Ile1
58417PRTHomo sapiensAntibody 14 84Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly8512PRTHomo
sapiensAntibody 14 85Trp Ala Asp Asp His Asn Tyr Pro His Ile Asp
Val1 5 1086324DNAHomo sapiensAntibody 14 86gacatcgtga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggtattagt agctggttgg cctggtatca gcagaaacca 120gggagagccc
ctaaggtctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg caacttacta ctgtgccgcc cactacgccg
ccccgtggac gttcggccaa 300gggaccaagc tggagatcaa acgt 32487108PRTHomo
sapiensAntibody 14 87Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Ala Ala His Tyr Ala Ala Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg 100 1058811PRTHomo sapiensAntibody
14 88Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 10897PRTHomo
sapiensAntibody 14 89Lys Ala Ser Thr Leu Glu Ser1 5909PRTHomo
sapiensAntibody 14 90Ala Ala His Tyr Ala Ala Pro Trp Thr1
591360DNAHomo sapiensAntibody 16 91gaagtgcagc tggtgcagtc tgggggaggc
ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt caccatcagc
agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg ggctggagtg
ggtctccgat ctttattatt atgctggtga cacatattac 180gcagactctg
tgaagggccg attcaccatg tccagagaca tttccaagaa caccgtgtat
240cttcaaatga acagcctgag agccgaggac acgggtgtct attattgtgc
gagatgggcc 300gatgaccacc ccccctacat cgacctgtgg ggcaggggca
ccctggtcac cgtctcgagt 36092120PRTHomo sapiensAntibody 16 92Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25
30Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Pro Pro Tyr Ile
Asp Leu Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120935PRTHomo sapiensAntibody 16 93Ser Asn Tyr Met Ile1
59417PRTHomo sapiensAntibody 16 94Asp Leu Tyr Tyr Tyr Ala Gly Asp
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly9511PRTHomo
sapiensAntibody 16 95Trp Ala Asp Asp His Pro Pro Tyr Ile Asp Leu1 5
1096321DNAHomo sapiensAntibody 16 96gacatcgtga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggtattagt agctggttgg cctggtatca gcagaaacca 120gggagagccc
ctaaggtctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttggctcg
gcgggtcgtt cggccaaggg 300accaagctgg agatcaaacg t 32197107PRTHomo
sapiensAntibody 16 97Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90 95Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 1059811PRTHomo sapiensAntibody 16
98Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 10997PRTHomo
sapiensAntibody 16 99Lys Ala Ser Thr Leu Glu Ser1 51008PRTHomo
sapiensAntibody 16 100Gln Gln Ser Trp Leu Gly Gly Ser1
5101360DNAHomo sapiensAntibody 17 101gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt
caccatcagc agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg
ggctggagtg ggtctccgat ctttattatt atgctggtga cacatattac
180gcagactccg tgaagggccg attcaccatg tccagagaca tttccaagaa
caccgtgtat 240cttcaaatga acagcctgag agccgaggac acggctgtct
attattgtgc gagatgggcc 300gatgaccacc ccccctacat cgacatgtgg
ggcaggggca ccctggtcac cgtctcctca 360102120PRTHomo sapiensAntibody
17 102Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser
Ser Asn 20 25 30Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser
Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Pro
Pro Tyr Ile Asp Met Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val
Ser Ser 115 1201035PRTHomo sapiensAntibody 17 103Ser Asn Tyr Met
Ile1 510417PRTHomo sapiensAntibody 17 104Asp Leu Tyr Tyr Tyr Ala
Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly10511PRTHomo
sapiensAntibody 17 105Trp Ala Asp Asp His Pro Pro Tyr Ile Asp Met1
5 10106321DNAHomo sapiensAntibody 17 106gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggtattagt agctggttgg cctggtatca gcagaaacca 120gggaaagccc
ctaaggtctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
tctgcaacct 240gatgattttg caacttacta ctgtcaacag agttggctcg
gcgggtcgtt cggccaaggg 300accaagctgg agatcaaacg t 321107107PRTHomo
sapiensAntibody 17 107Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90 95Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 10510811PRTHomo sapiensAntibody 17
108Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 101097PRTHomo
sapiensAntibody 17 109Lys Ala Ser Thr Leu Glu Ser1 51108PRTHomo
sapiensAntibody 17 110Gln Gln Ser Trp Leu Gly Gly Ser1
5111360DNAHomo sapiensAntibody 18 111gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc cgggggggtc cctgagactc 60tcctgtgcag
cctctgggtt caccatcagc agcaactaca tgatttgggt ccgtcaggct
120ccagggaagg ggctggagtg ggtctccgat ctttattatt atgctggtga
cacatattac 180gcagactccg tgaagggccg attcaccatg tccagagaca
tttccaagaa caccgtgtat 240cttcaaatga acagcctgag agccgaggac
acggctgtct attattgtgc gagatgggcc 300gatgaccacc ccccctggat
cgacctctgg ggcaggggca ccctggtcac cgtctcctca 360112120PRTHomo
sapiensAntibody 18 112Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Ile Ser Ser Asn 20 25 30Tyr Met Ile Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly
Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Met Ser
Arg Asp Ile Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala
Asp Asp His Pro Pro Trp Ile Asp Leu Trp Gly Arg 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 1201135PRTHomo sapiensAntibody 18
113Ser Asn Tyr Met Ile1 511417PRTHomo sapiensAntibody 18 114Asp Leu
Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly11511PRTHomo sapiensAntibody 18 115Trp Ala Asp Asp His Pro Pro
Trp Ile Asp Leu1 5 10116321DNAHomo sapiensAntibody 18 116gacatccaga
tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggccagtca gggtattagt agctggttgg cctggtatca gcagaaacca
120gggaaagccc ctaaggtctt gatctataag gcatctactt tagaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca
ccatcagcag tctgcaacct 240gatgattttg caacttacta ctgtcaacag
agttggctcg gcgggtcgtt cggccaaggg 300accaagctgg agatcaaacg t
321117107PRTHomo sapiensAntibody 18 117Asp Ile Gln Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser
Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90
95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 10511811PRTHomo
sapiensAntibody 18 118Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1
5 101197PRTHomo sapiensAntibody 18 119Lys Ala Ser Thr Leu Glu Ser1
51208PRTHomo sapiensAntibody 18 120Gln Gln Ser Trp Leu Gly Gly Ser1
5121360DNAHomo sapiensAntibody 19 121gaagtgcagc tggtgcagtc
tgggggaggc ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt
caccatcagc agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg
ggctggagtg ggtctccgat ctttattatt atgctggtga cacatattac
180gcagactccg tgaagggccg attcaccatg tccagagaca tttccaagaa
caccgtgtat 240cttcaaatga acagcctgag agccgaggac acgggtgtct
attattgtgc gagatgggcc 300gatgaccacc cctcccacct cgacatctgg
ggcaggggca ccctggtcac cgtctcgagt 360122120PRTHomo sapiensAntibody
19 122Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser
Ser Asn 20 25 30Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser
Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Pro
Ser His Leu Asp Ile Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val
Ser Ser 115 1201235PRTHomo sapiensAntibody 19 123Ser Asn Tyr Met
Ile1 512417PRTHomo sapiensAntibody 19 124Asp Leu Tyr Tyr Tyr Ala
Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly12511PRTHomo
sapiensAntibody 19 125Trp Ala Asp Asp His Pro Ser His Leu Asp Ile1
5 10126321DNAHomo sapiensAntibody 19 126gacatcgtga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggtattagt agctggttgg cctggtatca gcagaaacca 120gggagagccc
ctaaggtctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttggctcg
gcgggtcgtt cggccaaggg 300accaagctgg agatcaaacg t 321127107PRTHomo
sapiensAntibody 19 127Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90 95Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 10512811PRTHomo sapiensAntibody 19
128Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 101297PRTHomo
sapiensAntibody 19 129Lys Ala Ser Thr Leu Glu Ser1 51308PRTHomo
sapiensAntibody 19 130Gln Gln Ser Trp Leu Gly Gly Ser1
5131360DNAHomo sapiensAntibody 21 131gaagtgcagc tggtgcagtc
tgggggaggc ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt
caccatcagc agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg
ggctggagtg ggtctccgat ctttattatt atgctggtga cacatattac
180gcagactccg tgaagggccg attcaccatg tccagagaca tttccaagaa
caccgtgtat 240cttcaaatga acagcctgag agccgaggac acgggtgtct
attattgtgc gagatgggcc 300gatgaccacc cctcccacat tgacgtctgg
ggcaggggca ccctggtcac cgtctcgagt 360132120PRTHomo sapiensAntibody
21 132Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser
Ser Asn 20 25 30Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser
Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala Asp Asp His Pro
Ser His Ile Asp Val Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val
Ser Ser 115 1201335PRTHomo sapiensAntibody 21 133Ser Asn Tyr Met
Ile1 513417PRTHomo sapiensAntibody 21 134Asp Leu Tyr Tyr Tyr Ala
Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly13511PRTHomo
sapiensAntibody 21 135Trp Ala Asp Asp His Pro Ser His Ile Asp Val1
5 10136321DNAHomo sapiensAntibody 21 136gacatcgtga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gggtattagt agctggttgg cctggtatca gcagaaacca 120gggagagccc
ctaaggtctt gatctataag gcatctactt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttggctcg
gcgggtcgtt cggccaaggg 300accaagctgg agatcaaacg t 321137107PRTHomo
sapiensAntibody 21 137Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Arg Ala Pro Lys Val Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90 95Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 10513811PRTHomo sapiensAntibody 21
138Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 101397PRTHomo
sapiensAntibody 21 139Lys Ala Ser Thr Leu Glu Ser1 51408PRTHomo
sapiensAntibody 21 140Gln Gln Ser Trp Leu Gly Gly Ser1
5141363DNAHomo sapiensAntibody 22 141gaagtgcagc tggtgcagtc
tgggggaggc ttgatccagc cgggggggtc cctgagactc 60tcctgtgcag cctctgggtt
caccatcagc agcaactaca tgatttgggt ccgtcaggct 120ccagggaagg
ggctggagtg ggtctccgat ctttattatt atgctggtga cacatattac
180gcagactccg tgaagggccg attcaccatg tccagagaca tttccaagaa
caccgtgtat 240cttcaaatga acagcctgag agccgaggac acgggtgtct
attattgtgc gagatgggcc 300gatgaccaca acaacaccta cattgacgtc
tggggcaggg gcaccctggt caccgtctcg 360agt 363142121PRTHomo
sapiensAntibody 22 142Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu
Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Ile Ser Ser Asn 20 25 30Tyr Met Ile Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Asp Leu Tyr Tyr Tyr Ala Gly
Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Met Ser
Arg Asp Ile Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Arg Trp Ala
Asp Asp His Asn Asn Thr Tyr Ile Asp Val Trp Gly 100 105 110Arg Gly
Thr Leu Val Thr Val Ser Ser 115 1201435PRTHomo sapiensAntibody 22
143Ser Asn Tyr Met Ile1 514417PRTHomo sapiensAntibody 22 144Asp Leu
Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly14512PRTHomo sapiensAntibody 22 145Trp Ala Asp Asp His Asn Asn
Thr Tyr Ile Asp Val1 5 10146324DNAHomo sapiensAntibody 22
146gacatcgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc gggccagtca gggtattagt agctggttgg cctggtatca
gcagaaacca 120gggagagccc ctaaggtctt gatctataag gcatctactt
tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta
ctgtgccgcc cactacgccg ccccgtggac gttcggccaa 300gggaccaagc
tggagatcaa acgt 324147108PRTHomo sapiensAntibody 22 147Asp Ile Val
Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Val Leu Ile 35 40
45Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Ala His Tyr Ala
Ala Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 10514811PRTHomo sapiensAntibody 22 148Arg Ala Ser Gln Gly Ile
Ser Ser Trp Leu Ala1 5 101497PRTHomo sapiensAntibody 22 149Lys Ala
Ser Thr Leu Glu Ser1 51509PRTHomo sapiensAntibody 22 150Ala Ala His
Tyr Ala Ala Pro Trp Thr1 5151360DNAHomo sapiensAntibody 23
151gaagtgcagc tggtgcagtc tgggggaggc ttgatccagc cgggggggtc
cctgagactc 60tcctgtgcag cctctgggtt caccatcagc agcaactaca tgatttgggt
ccgtcaggct 120ccagggaagg ggctggagtg ggtctccgat ctttattatt
atgctggtga cacatattac 180gcagactccg tgaagggccg attcaccatg
tccagagaca tttccaagaa caccgtgtat 240cttcaaatga acagcctgag
agccgaggac acgggtgtct attattgtgc gagatgggcc 300gatgaccacg
ccccctgggt cgacctctgg ggcaggggca ccctggtcac cgtctcgagt
360152120PRTHomo sapiensAntibody 23 152Glu Val Gln Leu Val Gln Ser
Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25 30Tyr Met Ile Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Asp Leu Tyr
Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr Val Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Val Tyr Tyr Cys 85 90
95Ala Arg Trp Ala Asp Asp His Ala Pro Trp Val Asp Leu Trp Gly Arg
100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 1201535PRTHomo
sapiensAntibody 23 153Ser Asn Tyr Met Ile1 515417PRTHomo
sapiensAntibody 23 154Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr
Ala Asp Ser Val Lys1 5 10 15Gly15511PRTHomo sapiensAntibody 23
155Trp Ala Asp Asp His Ala Pro Trp Val Asp Leu1 5 10156321DNAHomo
sapiensAntibody 23 156gacatcgtga tgacccagtc tccttccacc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gggtattagt agctggttgg
cctggtatca gcagaaacca 120gggagagccc ctaaggtctt gatctataag
gcatctactt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcaacag agttggctcg gcgggtcgtt cggccaaggg
300accaagctgg agatcaaacg t 321157107PRTHomo sapiensAntibody 23
157Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Val
Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Trp Leu Gly Gly Ser 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg 100 10515811PRTHomo sapiensAntibody 23 158Arg Ala Ser Gln
Gly Ile Ser Ser Trp Leu Ala1 5 101597PRTHomo sapiensAntibody 23
159Lys Ala Ser Thr Leu Glu Ser1 51608PRTHomo sapiensAntibody 23
160Gln Gln Ser Trp Leu Gly Gly Ser1 5161212PRTHomo sapiensFull
length IL-6 161Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala
Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala
Pro Val Pro Pro 20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His
Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg
Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn
Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu
Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe
Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile
Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln
Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135
140Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys
Asn145 150 155 160Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn
Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu
Gln Asp Met Thr Thr His 180 185 190Leu Ile Leu Arg Ser Phe Lys Glu
Phe Leu Gln Ser
Ser Leu Arg Ala 195 200 205Leu Arg Gln Met 210162203PRTHomo
sapiensHIS FLAG tagged IL-6 162Met Gly Ser Ser His His His His His
His Asp Tyr Lys Asp Asp Asp1 5 10 15Asp Lys His Met Val Pro Pro Gly
Glu Asp Ser Lys Asp Val Ala Ala 20 25 30Pro His Arg Gln Pro Leu Thr
Ser Ser Glu Arg Ile Asp Lys Gln Ile 35 40 45Arg Tyr Ile Leu Asp Gly
Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn 50 55 60Lys Ser Asn Met Cys
Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn65 70 75 80Leu Asn Leu
Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly 85 90 95Phe Asn
Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu 100 105
110Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu
115 120 125Glu Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile
Gln Phe 130 135 140Leu Gln Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr
Thr Pro Asp Pro145 150 155 160Thr Thr Asn Ala Ser Leu Leu Thr Lys
Leu Gln Ala Gln Asn Gln Trp 165 170 175Leu Gln Asp Met Thr Thr His
Leu Ile Leu Arg Ser Phe Lys Glu Phe 180 185 190Leu Gln Ser Ser Leu
Arg Ala Leu Arg Gln Met 195 200163358PRTHomo sapiensSoluble IL-6Ra
163Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro1
5 10 15Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala
Arg 20 25 30Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr
Cys Pro 35 40 45Gly Val Glu Pro Glu Asp Asn Ala Thr Val His Trp Val
Leu Arg Lys 50 55 60Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala Gly
Met Gly Arg Arg65 70 75 80Leu Leu Leu Arg Ser Val Gln Leu His Asp
Ser Gly Asn Tyr Ser Cys 85 90 95Tyr Arg Ala Gly Arg Pro Ala Gly Thr
Val His Leu Leu Val Asp Val 100 105 110Pro Pro Glu Glu Pro Gln Leu
Ser Cys Phe Arg Lys Ser Pro Leu Ser 115 120 125Asn Val Val Cys Glu
Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr 130 135 140Lys Ala Val
Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp145 150 155
160Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys
165 170 175Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val
Ser Met 180 185 190Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys
Thr Gln Thr Phe 195 200 205Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro
Pro Ala Asn Ile Thr Val 210 215 220Thr Ala Val Ala Arg Asn Pro Arg
Trp Leu Ser Val Thr Trp Gln Asp225 230 235 240Pro His Ser Trp Asn
Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg 245 250 255Tyr Arg Ala
Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp 260 265 270Leu
Gln His His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His 275 280
285Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser
290 295 300Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser
Arg Ser305 310 315 320Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Met
Gln Ala Leu Thr Thr 325 330 335Asn Lys Asp Asp Asp Asn Ile Leu Phe
Arg Asp Ser Ala Asn Ala Thr 340 345 350Ser Leu Pro Val Gln Asp
355164468PRTHomo sapiensTransmembrane IL-6Ra 164Met Leu Ala Val Gly
Cys Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro1 5 10 15Gly Ala Ala Leu
Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg 20 25 30Gly Val Leu
Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro 35 40 45Gly Val
Glu Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys 50 55 60Pro
Ala Ala Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg65 70 75
80Leu Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys
85 90 95Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp
Val 100 105 110Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser
Pro Leu Ser 115 120 125Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr
Pro Ser Leu Thr Thr 130 135 140Lys Ala Val Leu Leu Val Arg Lys Phe
Gln Asn Ser Pro Ala Glu Asp145 150 155 160Phe Gln Glu Pro Cys Gln
Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys 165 170 175Gln Leu Ala Val
Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met 180 185 190Cys Val
Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe 195 200
205Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val
210 215 220Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp
Gln Asp225 230 235 240Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu
Arg Phe Glu Leu Arg 245 250 255Tyr Arg Ala Glu Arg Ser Lys Thr Phe
Thr Thr Trp Met Val Lys Asp 260 265 270Leu Gln His His Cys Val Ile
His Asp Ala Trp Ser Gly Leu Arg His 275 280 285Val Val Gln Leu Arg
Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser 290 295 300Glu Trp Ser
Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser305 310 315
320Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr
325 330 335Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn
Ala Thr 340 345 350Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pro Leu
Pro Thr Phe Leu 355 360 365Val Ala Gly Gly Ser Leu Ala Phe Gly Thr
Leu Leu Cys Ile Ala Ile 370 375 380Val Leu Arg Phe Lys Lys Thr Trp
Lys Leu Arg Ala Leu Lys Glu Gly385 390 395 400Lys Thr Ser Met His
Pro Pro Tyr Ser Leu Gly Gln Leu Val Pro Glu 405 410 415Arg Pro Arg
Pro Thr Pro Val Leu Val Pro Leu Ile Ser Pro Pro Val 420 425 430Ser
Pro Ser Ser Leu Gly Ser Asp Asn Thr Ser Ser His Asn Arg Pro 435 440
445Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp Ile Ser Asn Thr Asp Tyr
450 455 460Phe Phe Pro Arg465165183PRTHomo sapiensMature IL-6
165Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln1
5 10 15Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile
Leu 20 25 30Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser
Asn Met 35 40 45Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu
Asn Leu Pro 50 55 60Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly
Phe Asn Glu Glu65 70 75 80Thr Cys Leu Val Lys Ile Ile Thr Gly Leu
Leu Glu Phe Glu Val Tyr 85 90 95Leu Glu Tyr Leu Gln Asn Arg Phe Glu
Ser Ser Glu Glu Gln Ala Arg 100 105 110Ala Val Gln Met Ser Thr Lys
Val Leu Ile Gln Phe Leu Gln Lys Lys 115 120 125Ala Lys Asn Leu Asp
Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala 130 135 140Ser Leu Leu
Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met145 150 155
160Thr Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser
165 170 175Leu Arg Ala Leu Arg Gln Met 180166918PRTHomo
sapiensHuman gp130 166Met Leu Thr Leu Gln Thr Trp Leu Val Gln Ala
Leu Phe Ile Phe Leu1 5 10 15Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp
Pro Cys Gly Tyr Ile Ser 20 25 30Pro Glu Ser Pro Val Val Gln Leu His
Ser Asn Phe Thr Ala Val Cys 35 40 45Val Leu Lys Glu Lys Cys Met Asp
Tyr Phe His Val Asn Ala Asn Tyr 50 55 60Ile Val Trp Lys Thr Asn His
Phe Thr Ile Pro Lys Glu Gln Tyr Thr65 70 75 80Ile Ile Asn Arg Thr
Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser 85 90 95Leu Asn Ile Gln
Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu 100 105 110Gln Asn
Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys 115 120
125Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Arg Cys
130 135 140Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr Asn Phe
Thr Leu145 150 155 160Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp
Cys Lys Ala Lys Arg 165 170 175Asp Thr Pro Thr Ser Cys Thr Val Asp
Tyr Ser Thr Val Tyr Phe Val 180 185 190Asn Ile Glu Val Trp Val Glu
Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205Ser Asp His Ile Asn
Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220Pro His Asn
Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu225 230 235
240Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys
245 250 255Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser
Gln Ile 260 265 270Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe
Thr Val Gln Asp 275 280 285Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg
Ile Arg Cys Met Lys Glu 290 295 300Asp Gly Lys Gly Tyr Trp Ser Asp
Trp Ser Glu Glu Ala Ser Gly Ile305 310 315 320Thr Tyr Glu Asp Arg
Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile 325 330 335Asp Pro Ser
His Thr Gln Gly Tyr Arg Thr Val Gln Leu Val Trp Lys 340 345 350Thr
Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val 355 360
365Thr Leu Thr Arg Trp Lys Ser His Leu Gln Asn Tyr Thr Val Asn Ala
370 375 380Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala
Thr Leu385 390 395 400Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala
Ala Val Leu Thr Ile 405 410 415Pro Ala Cys Asp Phe Gln Ala Thr His
Pro Val Met Asp Leu Lys Ala 420 425 430Phe Pro Lys Asp Asn Met Leu
Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445Ser Val Lys Lys Tyr
Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460Pro Cys Ile
Thr Asp Trp Gln Gln Glu Asp Gly Thr Val His Arg Thr465 470 475
480Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val
485 490 495Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile
Lys Ala 500 505 510Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr
Val Arg Thr Lys 515 520 525Lys Val Gly Lys Asn Glu Ala Val Leu Glu
Trp Asp Gln Leu Pro Val 530 535 540Asp Val Gln Asn Gly Phe Ile Arg
Asn Tyr Thr Ile Phe Tyr Arg Thr545 550 555 560Ile Ile Gly Asn Glu
Thr Ala Val Asn Val Asp Ser Ser His Thr Glu 565 570 575Tyr Thr Leu
Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590Ala
Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600
605Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu Ala Ile Val Val Pro
610 615 620Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu
Phe Cys625 630 635 640Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile
Trp Pro Asn Val Pro 645 650 655Asp Pro Ser Lys Ser His Ile Ala Gln
Trp Ser Pro His Thr Pro Pro 660 665 670Arg His Asn Phe Asn Ser Lys
Asp Gln Met Tyr Ser Asp Gly Asn Phe 675 680 685Thr Asp Val Ser Val
Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700Pro Glu Asp
Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn705 710 715
720Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser
725 730 735Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser
Gln Asn 740 745 750Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His
Ser Gly Tyr Arg 755 760 765His Gln Val Pro Ser Val Gln Val Phe Ser
Arg Ser Glu Ser Thr Gln 770 775 780Pro Leu Leu Asp Ser Glu Glu Arg
Pro Glu Asp Leu Gln Leu Val Asp785 790 795 800His Val Asp Gly Gly
Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys 805 810 815Gln Asn Cys
Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu 820 825 830Arg
Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840
845Lys Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln
850 855 860Met Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly
Pro Gly865 870 875 880Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val
Gly Met Glu Ala Ala 885 890 895Thr Asp Glu Gly Met Pro Lys Ser Tyr
Leu Pro Gln Thr Val Arg Gln 900 905 910Gly Gly Tyr Met Pro Gln
91516730PRTHomo sapiensVh3_DP-86_(3-66) FW1 167Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser 20 25 3016814PRTHomo
sapiensVh3_DP-86_(3-66) FW2 168Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser1 5 1016932PRTHomo sapiensVh3_DP-86_(3-66) FW3
169Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr Val Tyr Leu Gln1
5 10 15Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg 20 25 3017011PRTHomo sapiensVh3_DP-86_(3-66) FW4 170Trp Gly Arg
Gly Thr Leu Val Thr Val Ser Ser1 5 1017123PRTHomo sapiensVk1_L12
FW1 171Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys 2017215PRTHomo
sapiensVk1_L12 FW2 172Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Val Leu Ile Tyr1 5 10 1517332PRTHomo sapiensVk1_L12 FW3 173Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr1 5 10 15Leu
Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys 20 25
3017411PRTHomo sapiensVk1_L12 FW4 174Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg1 5 10175212PRTHomo sapiensPhe102Glu mutant full
length IL-6 175Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala
Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala
Pro Val Pro Pro 20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His
Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg
Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn
Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu
Asn Asn Leu Asn Leu Pro Lys Met Ala 85
90 95Glu Lys Asp Gly Cys Glu Gln Ser Gly Phe Asn Glu Glu Thr Cys
Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr
Leu Glu Tyr 115 120 125Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln
Ala Arg Ala Val Gln 130 135 140Met Ser Thr Lys Val Leu Ile Gln Phe
Leu Gln Lys Lys Ala Lys Asn145 150 155 160Leu Asp Ala Ile Thr Thr
Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln
Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190Leu Ile
Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala 195 200
205Leu Arg Gln Met 210176212PRTHomo sapiensSer204Glu mutant full
length IL-6 176Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala
Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala
Pro Val Pro Pro 20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His
Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg
Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn
Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu
Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe
Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile
Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln
Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135
140Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys
Asn145 150 155 160Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn
Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu
Gln Asp Met Thr Thr His 180 185 190Leu Ile Leu Arg Ser Phe Lys Glu
Phe Leu Gln Glu Ser Leu Arg Ala 195 200 205Leu Arg Gln Met
210177212PRTHomo sapiensArg207Glu mutant full length IL-6 177Met
Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu1 5 10
15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro
20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu
Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp
Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met
Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn
Leu Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe
Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu
Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln Asn Arg Phe Glu
Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135 140Met Ser Thr Lys
Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn145 150 155 160Leu
Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170
175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His
180 185 190Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu
Glu Ala 195 200 205Leu Arg Gln Met 210178212PRTHomo
sapiensPhe106Glu mutant full length IL-6 178Met Asn Ser Phe Ser Thr
Ser Ala Phe Gly Pro Val Ala Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val
Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30Gly Glu Asp Ser
Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu
Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala
Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser65 70 75
80Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala
85 90 95Glu Lys Asp Gly Cys Phe Gln Ser Gly Glu Asn Glu Glu Thr Cys
Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr
Leu Glu Tyr 115 120 125Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln
Ala Arg Ala Val Gln 130 135 140Met Ser Thr Lys Val Leu Ile Gln Phe
Leu Gln Lys Lys Ala Lys Asn145 150 155 160Leu Asp Ala Ile Thr Thr
Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln
Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190Leu Ile
Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala 195 200
205Leu Arg Gln Met 210179212PRTHomo sapiensGln211Ala mutant full
length IL-6 179Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala
Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala
Pro Val Pro Pro 20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His
Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg
Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn
Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu
Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe
Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile
Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln
Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135
140Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys
Asn145 150 155 160Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn
Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu
Gln Asp Met Thr Thr His 180 185 190Leu Ile Leu Arg Ser Phe Lys Glu
Phe Leu Gln Ser Ser Leu Arg Ala 195 200 205Leu Arg Ala Met
210180212PRTHomo sapiensArg58Glu mutant full length IL-6 180Met Asn
Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu1 5 10 15Gly
Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25
30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr
35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Glu Tyr Ile Leu Asp Gly
Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys
Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu
Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn
Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu Glu
Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln Asn Arg Phe Glu Ser
Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135 140Met Ser Thr Lys Val
Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn145 150 155 160Leu Asp
Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170
175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His
180 185 190Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu
Arg Ala 195 200 205Leu Arg Gln Met 210181212PRTHomo
sapiensGlu200Trp mutant full length IL-6 181Met Asn Ser Phe Ser Thr
Ser Ala Phe Gly Pro Val Ala Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val
Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30Gly Glu Asp Ser
Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu
Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala
Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser65 70 75
80Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala
85 90 95Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys
Leu 100 105 110Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr
Leu Glu Tyr 115 120 125Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln
Ala Arg Ala Val Gln 130 135 140Met Ser Thr Lys Val Leu Ile Gln Phe
Leu Gln Lys Lys Ala Lys Asn145 150 155 160Leu Asp Ala Ile Thr Thr
Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln
Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190Leu Ile
Leu Arg Ser Phe Lys Trp Phe Leu Gln Ser Ser Leu Arg Ala 195 200
205Leu Arg Gln Met 210182212PRTHomo sapiensArg207Leu mutant full
length IL-6 182Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala
Phe Ser Leu1 5 10 15Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala
Pro Val Pro Pro 20 25 30Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His
Arg Gln Pro Leu Thr 35 40 45Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg
Tyr Ile Leu Asp Gly Ile 50 55 60Ser Ala Leu Arg Lys Glu Thr Cys Asn
Lys Ser Asn Met Cys Glu Ser65 70 75 80Ser Lys Glu Ala Leu Ala Glu
Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95Glu Lys Asp Gly Cys Phe
Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110Val Lys Ile Ile
Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125Leu Gln
Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135
140Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys
Asn145 150 155 160Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn
Ala Ser Leu Leu 165 170 175Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu
Gln Asp Met Thr Thr His 180 185 190Leu Ile Leu Arg Ser Phe Lys Glu
Phe Leu Gln Ser Ser Leu Leu Ala 195 200 205Leu Arg Gln Met
21018333DNAMacaca fascicularisPrimer macIL6_5'NdeI 183ttatcatatg
gtactcccag gagaagattc caa 3318430DNAMacaca fascicularisPrimer
macIL6_3'NheI 184ttatgctagc ctacatttgc cgaagagccc 3018532DNAHomo
sapiensPrimer hIL6_5'NdeI 185ttatacatat ggtaccccca ggagaagatt cc
32
* * * * *
References