U.S. patent application number 13/606642 was filed with the patent office on 2013-02-14 for anti-human interleukin-20 antibodies.
This patent application is currently assigned to Novo Nordisk A/S. The applicant listed for this patent is Jes Thorn Clausen, Soren Ostergaard, Jesper Pass. Invention is credited to Jes Thorn Clausen, Soren Ostergaard, Jesper Pass.
Application Number | 20130039923 13/606642 |
Document ID | / |
Family ID | 40947599 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130039923 |
Kind Code |
A1 |
Pass; Jesper ; et
al. |
February 14, 2013 |
ANTI-HUMAN INTERLEUKIN-20 ANTIBODIES
Abstract
Anti-human IL20 monoclonal antibodies that can reduce IL20
mediated activation of both IL20R1/IL20R2 and IL22R1/IL20R2
receptor complexes in one or more species, including humans, are
described, as well as antigen-binding molecules such as, e.g.,
antigen-binding antibody fragments, antibody derivatives, and
multi-specific molecules designed or derived from such antibodies,
and methods or producing such antibodies or other antigen-binding
molecules. Such antibodies or other antigen-binding molecules can
be used for treating various diseases and disorders, including
autoimmune or inflammatory diseases or disorders.
Inventors: |
Pass; Jesper; (Alleroed,
DK) ; Ostergaard; Soren; (Broenshoej, DK) ;
Clausen; Jes Thorn; (Hoeng, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pass; Jesper
Ostergaard; Soren
Clausen; Jes Thorn |
Alleroed
Broenshoej
Hoeng |
|
DK
DK
DK |
|
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
40947599 |
Appl. No.: |
13/606642 |
Filed: |
September 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12601164 |
Jan 7, 2010 |
8287861 |
|
|
PCT/EP2009/058155 |
Jun 30, 2009 |
|
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13606642 |
|
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61079005 |
Jul 8, 2008 |
|
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Current U.S.
Class: |
424/158.1 ;
435/69.6; 530/387.9; 530/389.2 |
Current CPC
Class: |
A61P 37/02 20180101;
C07K 16/244 20130101; C07K 2317/21 20130101; A61P 1/04 20180101;
A61P 37/06 20180101; C07K 2317/56 20130101; C07K 2317/73 20130101;
A61P 29/00 20180101; A61P 1/00 20180101; C07K 2317/34 20130101;
A61P 17/06 20180101; C07K 2317/76 20130101; A61P 19/02 20180101;
A61P 25/00 20180101; A61P 37/00 20180101; C07K 2317/565 20130101;
A61P 13/12 20180101 |
Class at
Publication: |
424/158.1 ;
530/389.2; 530/387.9; 435/69.6 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61P 29/00 20060101 A61P029/00; A61P 19/02 20060101
A61P019/02; C12P 21/02 20060101 C12P021/02; A61P 25/00 20060101
A61P025/00; A61P 1/00 20060101 A61P001/00; A61P 13/12 20060101
A61P013/12; A61P 37/00 20060101 A61P037/00; A61K 39/395 20060101
A61K039/395; A61P 17/06 20060101 A61P017/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2008 |
EP |
08159344.4 |
Claims
1. An isolated anti-human IL20 antibody or an antigen-binding
fragment thereof comprising a heavy chain variable region that is
derived from a set of human genes comprising VH1.sub.--03, D3-10,
and JH6 genes.
2. The antibody or antigen-binding fragment of claim 1, wherein the
heavy chain variable region comprises the CDR2 and CDR3 sequences,
and optionally the CDR1 sequence, of SEQ ID NO:8, respectively
corresponding to Kabat residues 50-65, 95-102, and 31-35.
3. The antibody or antigen-binding fragment of claim 1, comprising
a light chain variable region that is derived from a set of human
genes comprising VKI_L18 and JK4 genes.
4. The antibody or antigen-binding fragment of claim 1, comprising
the light chain variable sequence of SEQ ID NO:9 and the
heavy-chain variable sequence of SEQ ID NO:6 or SEQ ID NO:7.
5. A human antibody, or an antigen-binding fragment thereof, which
binds to human IL20 and has one or more properties selected from
(a) reduces IL20-mediated activation of IL20R1/IL20R2 and
IL22R1/IL20R2 receptor complexes; (b) reduces IL20-mediated
proliferation of BaF-3 cells recombinantly expressing
IL20R1/IL20R2; (c) does not reduce IL19- or IL24-mediated
proliferation of BaF-3 cells recombinantly expressing
IL20R1/IL20R2; (d) binds to human IL20 with a KD of about 1 nM or
less; and (e) has a solubility of at least about 80 mg/ml in an
aqueous buffered solution at about pH 7.4.
6. The antibody or antigen-binding fragment of claim 5, which
competes in binding to human IL20 with an antibody comprising a
light-chain variable region comprising SEQ ID NO:9 and a
heavy-chain variable region comprising SEQ ID NO:6 or SEQ ID
NO:7.
7. The antibody or antigen-binding fragment of claim 6, which binds
to an epitope comprising at least one residue selected from
H79-H103 of mature human IL20 (SEQ ID NO:1).
8. The antibody or antigen-binding fragment of claim 7, wherein the
epitope comprises at least one residue selected from H79-L93.
9. The antibody or antigen-binding fragment of claim 5, comprising
a heavy chain variable region comprising CDR1, CDR2, and CDR3
sequences comprising Kabat residues residues 31-35, 50-65, and
95-102, respectively, of SEQ ID NO:6 or SEQ ID NO:7.
10. The antibody or antigen-binding fragment of claim 9, comprising
a light chain variable region comprising CDR1, CDR2, and CDR3
sequences comprising Kabat residues 24-34, 50-56 and 89-97,
respectively, of SEQ ID NO:9.
11. The antibody of claim 1, which is of the IgG4 isotype.
12. A pharmaceutical composition comprising at least about 80 mg/ml
of the antibody of claim 1, and a pharmaceutically acceptable
excipient, diluent, or carrier.
13. The antibody, antigen-binding fragment, or pharmaceutical
composition of claim 1 for use in treating an inflammatory or
autoimmune disorder.
14. The antibody, antigen-binding fragment, or pharmaceutical
composition of claim 13, for use in treating rheumatoid arthritis,
juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis,
ankylosing spondylitis, Sjogren's syndrome, multiple sclerosis,
inflammatory bowel disease, systemic lupus erythematosus, lupus
nephritis, or a combination thereof.
15. A method of producing an anti-IL20 antibody or antigen-binding
fragment, comprising culturing a host cell producing the antibody
or antigen-binding fragment of claim 1 under suitable conditions,
and recovering said antibody or antigen-binding fragment.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/601,164, filed Nov. 20, 2009, which is a 371 National Stage
Filing of International Application No. PCT/EP2009/058155, filed
Jun. 30, 2009, which claims priority to European Application No.
08159344.4, filed Jun. 30, 2008, and which also claims priority to
U.S. Application No. 61/079,005, filed Jul. 8, 2008; the contents
of all above-named applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies against human
interleukin-20 (IL20), including human monoclonal anti-IL20
antibodies, as well as methods of production, compositions, and use
thereof.
BACKGROUND OF THE INVENTION
[0003] Interleukin-19 (IL19), IL20, and interleukin-24 (IL24) are
members of the interleukin-10 (IL10) cytokine family. All three
interleukins bind and signal through the IL20R1/IL20R2
heterodimeric receptor. IL20 and IL24 (but not IL19) are also
ligands for the receptor complex composed of IL20R2 and IL22R1
(Parrish-Novak et al., J Biol Chem 2002; 277: 47517-47523;
Dumoutier et al., J Immunol 2001; 167:3545-3549). It has been
proposed that IL19 and IL20, along with other IL10 family members,
form a distinct subfamily of helical cytokines where at least IL19
and IL20 have similar three-dimensional structures (Chang et al., J
Biol Chem 2003; 278: 3308-13).
[0004] IL20 and its receptors are present in elevated levels in
psoriatic lesions (Wei et al., Clin Immunol (2005) 117: 65-72;
Romer et al., J Invest Dermatol 2003; 121, 1306-1311; Wang et al.,
J Invest Dermatol 2006; 126: 1590-1599; Otkjr et al., Br J Dermatol
2005; 153: 911-918) and in synovial fluid of rheumatoid arthritis
patients (Hsu et al., Arthritis Rheum 2006; 54: 2722-2733;
Kragstrup et al., Cytokine 2008; 41: 16-2). Antagonizing IL20
activity using receptor fragments or monoclonal antibodies has
therefore been described as a promising approach for treatment of
various inflammatory conditions (e.g., WO9927103, WO0146261,
WO2003051384, WO2004085475, and WO2006086396). For example,
polyclonal anti-IL20 antibodies were found to be therapeutically
effective in a xenograft model of psoriasis (Stenderup et al., Br J
Dermatol 2006; 154: 11-35, Abstract P-12; Stenderup et al. Br J
Dermatol 2009; 160(2):284-96).
[0005] Antigenic epitopes of human IL20 (hIL20), as well as rat or
murine monoclonal antibodies binding hulL20, have also been
described (e.g., WO2005052000, US20060142550, and WO2007081465).
However, no antibodies suitable for patient treatment have so far
been provided. The present invention addresses these and other
needs in the art.
SUMMARY OF THE INVENTION
[0006] The present invention provides anti-hIL20 monoclonal
antibodies that can reduce IL20-mediated activation of
IL20R1/IL20R2 and IL22R1/IL20R2 receptor complexes in one or more
species, including humans. Typically, the antibodies are fully
human or humanized to minimize the risk for immune responses
against the antibodies when administered to a patient. The
invention also provides anti-hIL20 antibodies having improved
solubility properties, making them capable of being formulated at
high concentrations. As described herein, other antigen-binding
molecules such as, e.g., antigen-binding antibody fragments,
antibody derivatives, and multi-specific molecules, can be designed
or derived from such antibodies.
[0007] Antibodies binding a specific segment of the hIL20 molecule
that corresponds to Helix E in IL19 are also provided. In one
embodiment, the epitope of the antibody comprise one or more amino
acid residues in the segment corresponding to D78-L93, optionally
excluding D78, in mature hIL20 (SEQ ID NO:1), e.g., H79, R83, S85,
N90, F92, L93, or any combination thereof.
[0008] Certain anti-hIL20 antibodies of the invention may also
compete with and/or bind to the same epitope or have the same
binding interface on hIL20 as one or more of the specific human
anti-hIL20 antibodies described herein, including 15D2 and 5B7. For
example, in one embodiment, the antibodies of the invention are
more capable of competing with 15D2 and/or 5B7 than with known
anti-hIL20 antibodies.
[0009] In another aspect, antibodies of the invention comprise
antigen-binding sequences that derive from one or more of the same
human V, D, or J segments as 15D2 or 5B7. The antibodies may, for
example, comprise one or more antigen-binding sequences that are
identical or substantially identical to 15D2 and/or 5B7
antigen-binding sequences described herein.
[0010] In other aspects, the invention provides for nucleic acids
encoding antibodies of the invention, expression vectors comprising
such nucleic acids, host cells comprising such nucleic acids, host
cells producing antibodies of the invention, and methods of
producing anti-hIL20 antibodies by culturing such host cells under
appropriate conditions. Also provided for are antibody-binding
fragments of such antibodies, and molecules comprising such
antibodies or antigen-binding fragments, including engineered
antibody fragments, antibody derivatives, bispecific antibodies and
other multispecific molecules. Pharmaceutical compositions and kits
or other articles that comprise such antibodies or molecules can
also be prepared. Further provided for are methods of reducing or
inhibiting IL20-mediated activation of IL20R1/IL20R2 and
IL22R1/IL20R2 receptor complexes, and methods of treating or
preventing autoimmune or inflammatory diseases or disorders,
including, but not limited to rheumatoid arthritis, juvenile
rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing
spondylitis, Sjogren's syndrome, multiple sclerosis, inflammatory
bowel disease, systemic lupus erythematosus, lupus nephritis, or a
combination thereof, using such antibodies.
DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the amino acid sequence of mature hIL20 (A)
(SEQ ID NO:1) and (B) an alignment of the precursor form of hIL20
(SEQ ID NO:2), human IL19 (SEQ ID NO:3), murine IL20 (SEQ ID NO:4),
and cynomolgus IL20 (SEQ ID NO:5). In (A), the numbered residues
correspond to Helix E in IL19, with bold residues representing the
key epitope segment of 15D2 and 5B7. Bold underlined residues are
the ones most important for 15D2 and 5B7 binding, while bold
double-underlined residues were found to be critical for 15D2
and/or 5B7 binding. In (B), the underlined segment in hIL20 is the
signal sequence, and other markings are the same as in (A).
[0012] FIG. 2 shows a model of hIL20 (using IL19 nomenclature),
built using the Chemical Computing Group's Molecular Operating
Environment (MOE) software from the template 1N1F.pdb of hIL19.
Using a hIL19/hIL20 sequence alignment and the helix assignments in
1N1F.pdb, the figure was generated using Corel Draw (Corel
Corporation).
[0013] FIG. 3 shows analyses of 15D2 heavy-chain variable (VH)
region (SEQ ID NO:6) (A), 5B7 VH region (SEQ ID NO:7) (B), and
15D2/5B7 light-chain variable (VL) region (SEQ ID NO:9) (C), and an
alignment of the 15D2 and 5B7 VH regions along with a consensus
sequence (SEQ ID NO:8) (D). Each antibody sequence is aligned with
the corresponding germline sequences, showing the corresponding
Kabat-numbering of each amino acid position. In each sequence, the
corresponding complementarity-determining region (CDR) sequences
according to the Kabat scheme are shown in bold, underlined text.
VH1.sub.--03, D3-10, and JH6 correspond to sequences comprising SEQ
ID NOS:10, 12 and 14, respectively, and VKI_L18/JK4 correspond to
sequences comprising SEQ ID NOS:15 and 17, respectively. The coding
sequences of D3-10, JH6, and JK4 are provided in SEQ ID NOS: 11,
13, and 16, respectively.
[0014] FIG. 4 shows an alignment of VH (A) and VL (B) region
sequences of several human anti-IL20 antibodies of IgG4 isotype
with the corresponding Kabat-numbering of each amino acid position.
In each sequence, the corresponding CDR sequences according to the
Kabat scheme are shown in bold, underlined text. (A) Heavy chain
variable sequences for 2F6 (SEQ ID NO:18), C3 (SEQ ID NO:20), F18
(SEQ ID NO:22), F56 and F56/F18 (SEQ ID NO:23), 5B7 (SEQ ID NO:7),
and 15D2 (SEQ ID NO:6), (B) Light chain variable sequences for 2F6
(SEQ ID NO:19), C3 (SEQ ID NO:21), F56_type 1, 15D2, and 5B7 (SEQ
ID NO:9), and F56_type 2 (SEQ ID NO:24).
[0015] FIG. 5 shows the ability of 15D2 to inhibit hIL20-(A),
hIL19-(B), and hIL24-(C) induced proliferation of BaF-3 cells
transfected with hIL20R1/hIL20R2 at three different cytokine
concentrations. (A) A dose-dependent response was detected for
inhibition of hIL20-induced proliferation. No inhibition of
hIL19-(B) or hIL24-(C) induced proliferation was observed in
15D2-concentration range used (up to 66 nM).
[0016] FIG. 6 shows the results of a primary peptide array of hIL20
against 15D2 (A) or 5B7 (B). The Y axis indicates the optical
density (OD, a measure of fluorescence intensity). Note that not
all peptides are present in figure, since some OD values were below
detection limit. In (A), peptides corresponding to residues 69-86
(85), 73-90 (86), 77-94 (87), 81-98 (88), and 85-102 (89) of SEQ ID
NO:1 are shown. Peptide 87 came out as the peptide with highest
binding activity. In (B), peptides corresponding to residues 49-66
(19), 53-70 (20), 57-74 (21), 69-86 (24), 73-90 (25), and 77-94
(26) are shown.
[0017] FIG. 7 shows a secondary peptide array analysis of hIL20
against 15D2 (A) or 5B7 (B). The antibodies were tested against
constructs with truncations from the C- and N-terminal. The
peptides were all acylated in order to avoid the positive charge
arising from the N-terminal. In (A), peptides corresponding to Y74
to K96.fwdarw.S86 and K84 of SEQ ID NO:1 (peptides 1-12,
respectively) are shown on the left-hand column, and peptides
corresponding to Q75.fwdarw.I85 to K96 of SEQ ID NO:1 (peptides
13-24, respectively, where peptides 22 and 23 are identical) are
shown in the right-hand column. In (B), peptides corresponding to
Y74 to K96.fwdarw.S86 and K84 of SEQ ID NO:1 (peptides 1-12,
respectively) are shown on the left-hand column, and peptides
corresponding to Q75.fwdarw.S84 to K96 (peptides 13-24,
respectively, where peptides 22 and 23 are identical) are shown in
the right-hand column.
[0018] FIG. 8 shows an Ala-scan of the long epitope
YQTPDHYTLRKISSLANSFLTIK, corresponding to residues Y74 to K96 of
SEQ ID NO:1, against (A) 15D2 and (B) 5B7. In (A), the peptides
shown correspond to residues 78-96 of SEQ ID NO:1 with an alanine
substitution at positions 78-96, peptides 40-61, respectively. In
(B), the peptides shown correspond to residues 78-96 of SEQ ID NO:1
with an alanine substitution at positions 78-96, peptides 1-22,
respectively.
[0019] FIG. 9 shows 15D2 neutralization of murine IL20 activation
of murine IL22R1/IL20R2 receptor, as revealed by a luciferase
assay. Murine IL20 receptor complex mIL20R1/mIL22R1 was transfected
into BHK cells and stimulated with 10 nM murine IL20.
Neutralization of stimulation was investigated using 1
microgram/ml, 10 microgram/ml or 50 microgram/ml of 15D2.
[0020] FIG. 10 shows 15D2 neutralization of cynomolgus IL20
activation of human IL20R1/IL20R2 and IL22R1/IL20R2 receptors (A)
or cynomolgous IL20R1/IL20R2 and IL22R1/IL20R2 (B), as revealed by
a luciferase assay.
[0021] FIG. 11 shows 15D2 neutralization of human IL20-mediated
activation of human IL20R1/IL20R2 and IL22R1/IL20R2, as revealed by
a luciferase assay.
[0022] FIG. 12 shows that IL19 reverted the 15D2 blocking of
IL20-induced proliferation, revealing that 15D2 bound the soluble
form of IL20 and not the receptor-bound form, which would otherwise
block access of IL19 to the receptor.
[0023] FIG. 13 shows the primary sequence of hIL20 used in an amide
hydrogen/deuterium exchange (HX)-mass spectrometry (MS) study to
determine the 15D2 binding interface. The primary hIL20 sequence
(using mature Met-1 numbering, thus differing+1 from the
corresponding residue in SEQ ID NO:1) is displayed above the HX
analyzed peptides (shown as horizontal bars). Peptides showing
similar exchange patterns both in the presence and absence of 15D2
are indicated by grey bars whereas peptides showing reduced
deuterium incorporation upon 15D2 binding are indicated by black
bars.
[0024] FIG. 14 shows sub-localization of the deuterium label from
individual peptides in the HX-MS study. (A) Close-up of region
60-93 of the IL20 primary structure. Peptides showing similar
exchange patterns both in the presence and absence of 15D2 are
coloured grey whereas peptides showing reduced deuterium
incorporation upon 15D2 binding are coloured black. The numbers
indicate the difference in deuterium level observed in the
individual IL20 regions upon 15D2 binding. (B) The information from
the peptides have been sub-localized to smaller residue stretches
by simple subtraction assuming complete off-exchange of the
N-terminus and first peptide bond amide. The deuterium level was
then corrected for the labelling reaction only containing 91%
deuterium and reported as percent of total residues.
DEFINITIONS
[0025] Unless otherwise stated or contradicted by context, the
terms "IL20" or "IL-20" refer to human interleukin-20 (hIL20), also
known as Zcyto10, including its unprocessed precursor (UniProt
Q9NYY1; SEQ ID NO:2), mature form (SEQ ID NO:1, UniProt Q9NYY1
without the residues 1-24 signal sequence), and/or naturally
occurring variants or orthologs thereof, such as, e.g., murine IL20
(mIL20) precursor (UniProt Q9JKV9; SEQ ID NO:4), or cynomolgous
IL20 (cIL20) precursor (SEQ ID NO:5), or mature forms thereof which
lack the signal sequence corresponding to residues 1-24 in
precursor hIL20 (SEQ ID NO:2).
[0026] The term "antibody" herein is used in the broadest sense and
specifically includes full-length monoclonal antibodies, polyclonal
antibodies, and, unless otherwise stated or contradicted by
context, antigen-binding fragments, antibody variants, and
multispecific molecules thereof, so long as they exhibit the
desired specificity and/or biological activity. Generally, a
full-length antibody is a glycoprotein comprising at least two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, or an antigen binding portion thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as VH) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, CH1, CH2 and CH3.
Each light chain is comprised of a light chain variable region
(abbreviated herein as VL) and a light chain constant region. The
light chain constant region is comprised of one domain, CL. The VH
and VL regions can be further subdivided into regions of
hypervariability, termed complementarily determining regions
(abbreviated herein as CDR), interspersed with regions that are
more conserved, termed framework regions (FR). Each VH and VL is
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. Various
techniques relevant to the production of antibodies are provided
in, e.g., Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(1988).
[0027] An "antigen-binding fragment" of an antibody is a molecule
that comprises a portion of a full-length antibody which is capable
of detectably binding to the antigen. Antigen-binding fragments
include multivalent molecules comprising one, two, three, or more
antigen-binding portions of an antibody, and single-chain
constructs wherein the VL and VH regions, or selected portions
thereof, are joined by synthetic linkers or by recombinant methods
to form a functional, antigen-binding molecule.
[0028] The terms "antibody derivative" and immunoconjugate" are
used interchangeably herein to denote molecules comprising a
full-length antibody or an antigen-binding fragment thereof,
wherein one or more amino acids are chemically modified, e.g., by
alkylation, PEGylation, acylation, ester formation or amide
formation or the like, e.g., for linking the antibody to a second
molecule. Exemplary modifications include PEGylation,
cysteine-PEGylation, biotinylation, radiolabelling, and conjugation
with a second agent, such as a cytotoxic agent.
[0029] A "multispecific molecule" comprises an antibody, or an
antigen-binding fragment thereof, which is associated with or
linked to at least one other functional molecule (e.g. another
peptide or protein such as another antibody or ligand for a
receptor) to generate a molecule that binds to at least two
different binding sites or target molecules. Exemplary
multispecific molecules include bi-specific antibodies and
antibodies linked to soluble receptor fragments or ligands.
[0030] The term "human antibody", as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from human germline
immunoglobulin sequences. Furthermore, if the antibody contains a
constant region, the constant region also is derived from human
immunoglobulin sequences. Collections of human germline sequences
are available at, e.g., the NCBI website. The human antibodies of
the invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). However, the term "human antibody", as used herein, is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse,
have been grafted onto human framework sequences.
[0031] A "humanized" antibody is a human/non-human chimeric
antibody that contains a minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit, or non-human primate having the desired
specificity, affinity, and function. In some instances, FR residues
of the human immunoglobulin are replaced by corresponding non-human
residues ("back-mutations"). Furthermore, humanized antibodies may
comprise residues that are not found in the recipient antibody or
in the donor antibody. These modifications are made to further
refine antibody performance. In general, a humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR residues are
those of a human immunoglobulin sequence. The humanized antibody
can optionally also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see, e.g., Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), WO
92/02190, US Patent Application 20060073137, and U.S. Pat. Nos.
6,750,325, 6,632,927, 6,639,055, 6,548,640, 6,407,213, 6,180,370,
6,054,297, 5,929,212, 5,895,205, 5,886,152, 5,877,293, 5,869,619,
5,821,337, 5,821,123, 5,770,196, 5,777,085, 5,766,886, 5,714,350,
5,693,762, 5,693,761, 5,530,101, 5,585,089, and 5,225,539.
[0032] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region generally comprises amino
acid residues from a "complementarity-determining region" or "CDR"
(residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy-chain variable domain; (Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242) and/or
those residues from a "hypervariable loop" (residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light-chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable
domain; Chothia and Lesk, J. Mol. Biol. 1987; 196:901-917) and/or
the specificity-determining residues (SDRs), which are the residues
that are most crucial in the antibody-antigen interaction (Kashmiri
et al., Methods 2005; 36:25-34). The SDRs can be determined using,
e.g., 3D structural analysis of the antibody-antigen interaction or
by mutational analysis using known techniques. Typically, the
numbering of amino acid residues in this region is performed by the
method described in Kabat et al., supra. Phrases such as "Kabat
position", "variable domain residue numbering as in Kabat" and
"according to Kabat" herein refer to this numbering system for
heavy chain variable domains or light chain variable domains. Using
the Kabat numbering system, the actual linear amino acid sequence
of a peptide may contain fewer or additional amino acids
corresponding to a shortening of, or insertion into, a FR or CDR of
the variable domain. For example, a heavy chain variable domain may
include a single amino acid insert (residue 52a according to Kabat)
after residue 52 of CDR H2 and inserted residues (e.g. residues
82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR
residue 82. The Kabat numbering of residues may be determined for a
given antibody by alignment at regions of homology of the sequence
of the antibody with a "standard" Kabat numbered sequence (see
FIGS. 3 and 4).
[0033] A "variant" of a polypeptide refers to a polypeptide having
an amino acid sequence that is substantially identical to a
reference polypeptide, typically a native or "parent" polypeptide.
The polypeptide variant may possess one or more amino acid
substitutions, deletions, and/or insertions at certain positions
within the native amino acid sequence, but differs from the parent
polypeptide in at least one respect.
[0034] The term "epitope" or "antigenic determinant" of an antibody
is the part of a predetermined antigen to which the antibody binds,
and usually consists of chemically active surface groupings of
amino acids or sugar chains. The specific amino acids defining a
protein epitope can be relatively few in number, and typically
comprise the amino acids that are directly involved in binding to
the antibody, though other amino acids that are not directly
involved in binding to the antibody can nevertheless be blocked
when the antibody binds. The amino acids in a protein epitope may
be close to each other or widely dispersed along the length of
antigen, being brought into the correct epitope conformation via
folding. A "conformational epitope" refers to an epitope that
depends on the predetermined antigen being correctly folded, while
a "linear epitope" can also be recognized by the antibody when not
correctly folded, e.g., in denatured form or in the form of a
fragment comprising the epitope.
[0035] "Specific binding" as used herein refers to the ability of
an antibody to bind a predetermined antigen, such as, e.g., IL20.
Typically, the antibody binds with a dissociation constant (Kd) of
10.sup.-8 or less, and binds to the predetermined antigen with a Kd
that is at least 2-fold less than its Kd for binding to a
non-specific antigen (e.g., BSA) other than the predetermined
antigen or a closely related molecule (e.g., an ortholog).
[0036] The term "substantially identical" in the context of two
amino acid sequences means that the sequences, when optimally
aligned, such as by the programs GAP or BEST-FIT using default gap
weights, share at least about 50, at least about 60, at least about
70, at least about 80, at least about 90, at least about 95, at
least about 98, or at least about 99 percent sequence identity.
[0037] "Corresponding" amino acid positions in two substantially
identical amino acid sequences are those aligned by any of the
protein analysis software referred to herein, typically using
default parameters.
[0038] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a pre-sequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a pre-protein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome-binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0039] An "isolated" molecule is a molecule that is the predominant
species in the composition wherein it is found with respect to the
class of molecules to which it belongs (i.e., it makes up at least
about 50% of the type of molecule in the composition and typically
will make up at least about 70%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, or more of the species
of molecule, e.g., peptide, in the composition). Commonly, a
composition of an antibody molecule will exhibit 98%, 98%, or 99%
homogeneity for antibody molecules in the context of all present
peptide species in the composition or at least with respect to
substantially active peptide species in the context of proposed
use.
[0040] In the context of the present invention, "treatment" or
"treating" refers to preventing, alleviating, managing, curing or
reducing one or more symptoms or clinically relevant manifestations
of a disease or disorder, unless contradicted by context. For
example, "treatment" of a patient in whom no symptoms or clinically
relevant manifestations of a disease or disorder have been
identified is preventive or prophylactic therapy, whereas
"treatment" of a patient in whom symptoms or clinically relevant
manifestations of a disease or disorder have been identified
generally does not constitute preventive or prophylactic
therapy.
[0041] Unless otherwise expressly indicated or clearly contradicted
by context, the term "or" herein is used in the inclusive sense of
"and/or."
[0042] "Activation" of a receptor or receptor complex means an
increased or decreased activity of any or all intracellular signal
transduction elements associated with the receptor or receptor
complex after binding of the ligand to the receptor or receptor
complex under normal physiological or pathophysiological
conditions, as compared to a control. In the case of the
IL20R1/IL20R2 and IL22R1/IL20R2 receptor complexes, receptor
activation can be analyzed using, e.g., a luciferase assay similar
to the one described in Example 9. "Reducing activation" of a
receptor or receptor complex means that the activation of the
receptor is reduced by at least about 10%, preferably at least
about 20%, more preferably at least about 30%, most preferably at
least about 50%, or more, in comparison to a control (e.g., the
level of activation in the absence of antibody).
[0043] Some assays for evaluating the antibodies or other
antigen-binding molecules described herein employ one or more
"controls." A "control" may be a standard value retrieved from a
text book; a value obtained by running the same assay in the
absence of ligand (e.g., IL20), receptor (e.g., IL20R1/IL20R2
and/or IL22R1/IL20R2), or antibody; or in the presence of a
non-specific molecule (e.g., a non-specific antibody); or some
other reference value used in the art. In the case of a receptor
activation assay testing, for example, the ability of an antibody
to reduce activation of a receptor complex, a suitable control
value can be obtained by running the assay in the absence of the
antibody or in the presence of an antibody not specifically binding
to the ligand, receptor complex, or other components involved in
receptor activation.
DESCRIPTION OF THE INVENTION
[0044] The present invention provides for human anti-IL20
antibodies suitable for pharmaceutical formulations, diagnostic
uses, and therapeutic uses. As described in the Examples, a novel
epitope was identified for two human anti-IL20 antibodies
designated 15D2 and 5B7. Key epitope residues were mapped to a
region of hIL20 that corresponds to Helix E in hIL19 (Chang et al.,
J Biol Chem 2003; 278: 3308-13). A predicted model of hIL20 using
the hIL19 nomenclature is shown in FIG. 1, with Helix E
corresponding to residues D78 to H103 in the mature hIL20 sequence
(SEQ ID NO:1). It was found that an antibody binding to the novel
hIL20 epitope reduced hIL20-mediated activation of both the
IL20R1/IL20R2 and IL22R1/IL20R2 receptors, and reduced
IL20-mediated, but not IL19- or IL24-mediated, receptor activation
in a proliferation assay. The epitope was further found to exist in
both native and denatured form of the hIL20 antigen, as well as in
both murine and cynomolgous IL20. Both 15D2 and 5B7 were also
soluble at concentrations of at least about 80 mg/ml, and were
found to derive from the same set of human germline genes (Example
3 and FIG. 3).
[0045] The invention thus provides antibodies which combine one or
more functional properties, one or more structural properties,
and/or antibodies which combine one or more functional with one or
more structural properties described in subsequent sections and the
Examples.
[0046] In one aspect, the present invention provides an antibody,
such as a monoclonal human or humanized antibody, or an
antigen-binding fragment thereof, that specifically binds to hIL20,
optionally also to one or more hIL20 orthologs, specifically
reduces hIL20 mediated activation of both hIL20R1/hIL20R2 and
hIL22R1/hIL20R2 receptor complexes and/or their orthologs, and/or
has a high solubility. In one embodiment, the heavy or light chain
variable region sequences of the antibody derive from one or more
of the 15D2 and 5B7 germline and/or V, D, or J segments. In one
embodiment, the CDR and/or variable sequences of antibodies of the
invention are substantially identical to one or more
antigen-binding sequence of 15D2 and/or 5B7. In one embodiment, the
antibody interacts with one or more residues in the segment H79 to
H103 in the hIL20 sequence (SEQ ID NO:1), and may, for example,
bind to H79, R83, S85, N90, F91, L92, or a combination thereof. The
antibody may be in any form suitable for therapeutic applications,
e.g., a full-length antibody or a fragment thereof.
[0047] In one aspect, the invention provides for an isolated
anti-hIL20 antibody, or an antigen-binding fragment thereof,
comprising a heavy chain variable region that is derived from a set
of human genes comprising VH1.sub.--03, D3-10, and JH6 genes. In
one embodiment, the heavy chain variable region comprises the CDR2
and CDR3 sequences, and, optionally, the CDR1 sequence, of SEQ ID
NO:8, respectively corresponding to Kabat residues 50-65, 95-102,
and 31-35. In one embodiment, the light chain variable region
comprises a sequence derived from a set of human genes comprising
VKI_L18 and JK4 genes. In a specific embodiment, the light chain
variable region comprises the sequence of SEQ ID NO:9 and the
heavy-chain variable region comprises the sequence of SEQ ID NO:6
or SEQ ID NO:7. In another specific embodiment, the antibody is of
the IgG4 isotype.
[0048] In one aspect, the invention provides for a human antibody,
or an antigen-binding fragment thereof, which binds to hIL20 and
has one or more functional properties selected from (a) reducing
IL20-mediated activation of IL20R1/IL20R2 and IL22R1/IL20R2
receptor complexes; (b) reducing IL20-mediated proliferation of
BaF-3 cells recombinantly expressing IL20R1/IL20R2; (c) not
reducing IL19- or IL24-mediated proliferation of BaF-3 cells
recombinantly expressing IL20R1/IL20R2; (d) binding to hIL20 with a
KD of about 1 nM or less; and (e) has a solubility of at least
about 80 mg/ml in an aqueous buffered solution at about pH 7.4,
optionally comprising 150 mM NaCl. In one embodiment, the antibody
has properties (a) to (d). In one embodiment, the antibody has all
of properties (a) to (e). In one embodiment, the antibody or
antigen-binding fragment competes in binding to hIL20 with an
antibody comprising a light-chain variable region comprising SEQ ID
NO:9 and a heavy-chain variable region comprising SEQ ID NO:6 or
SEQ ID NO:7. In one embodiment, the antibody or antigen-binding
fragment binds to an epitope comprising at least one residue
selected from H79-H103 of mature hIL20 (SEQ ID NO:1). In one
embodiment, the epitope comprises at least one residue selected
from H79-L93. In one embodiment, the antibody or antigen-binding
fragment comprises a heavy chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising Kabat residues residues 31-35,
50-65, and 95-102, respectively, of SEQ ID NO:6 or SEQ ID NO:7. In
one embodiment, the antibody further comprises a light chain
variable region comprising CDR1, CDR2, and CDR3 sequences
comprising Kabat residues 24-34, 50-56 and 89-97, respectively, of
SEQ ID NO:9. In one embodiment, the antibody heavy and light chain
variable sequences are substantially identical to the respective
heavy and light chain variable sequences of 15D2 and/or 5B7, e.g.,
having a sequence identity of at least about 80%, at least about
90%, or at least about 95%. In a specific embodiment, the antibody
is of the IgG4 isotype.
[0049] In one aspect, the invention provides such human anti-hIL20
antibodies that are sufficiently soluble for use in pharmaceutical
compositions. In one embodiment, the invention provides a
pharmaceutical composition comprising an effective amount, e.g., at
a concentration of at least about 80 mg/ml or at least about 100
mg/ml, of an antibody of the invention, and a pharmaceutically
acceptable excipient, diluent, or carrier. In one embodiment, the
antibody is of an IgG4 isotype and comprises a heavy chain variable
region that is derived from a set of human genes comprising
VH1.sub.--03, D3-10, and JH6 genes and/or the light chain variable
region comprising a sequence derived from a set of human genes
comprising VKI_L18 and JK4 genes. In one specific embodiment, the
light chain variable region comprises the sequence of SEQ ID NO:9
and the heavy-chain variable region comprises the sequence of SEQ
ID NO:6. In one specific embodiment, the light chain variable
region comprises the sequence of SEQ ID NO:9 and the heavy-chain
variable region comprises the sequence of SEQ ID NO:7.
[0050] In one aspect, the invention provides for an antibody,
antigen-binding fragment, or pharmaceutical composition of the
invention for use as a medicament.
[0051] In one aspect, the invention provides for an antibody,
antigen-binding fragment, or pharmaceutical composition of the
invention for use in treating an inflammatory or autoimmune
disorder.
[0052] In one aspect, the invention provides for the use of an
antibody, antigen-binding fragment, or pharmaceutical composition
of the invention in the preparation of a medicament for treating an
inflammatory or autoimmune disorder.
[0053] In one aspect, the invention provides for a method of
treating a subject suffering from or at risk for an inflammatory or
autoimmune disorder by administering an antibody, antigen-binding
fragment, or pharmaceutical composition of the invention.
[0054] Inflammatory or autoimmune disorders suitable for such uses
include rheumatoid arthritis, juvenile rheumatoid arthritis,
psoriasis, psoriatic arthritis, ankylosing spondylitis, Sjogren's
syndrome, multiple sclerosis, inflammatory bowel disease, systemic
lupus erythematosus, or lupus nephritis, or a combination of any
thereof, as well as co-morbidities associated with these diseases,
with cardiovascular disease being a non-limiting example of said
co-morbidities.
[0055] In one aspect, the invention provides for a method of
recombinantly producing an anti-IL20 antibody or antigen-binding
fragment, comprising culturing a host cell producing the antibody
or antigen-binding fragment of the invention under suitable
conditions, and recovering the antibody or antigen-binding
fragment. The host cell typically comprises an expression vector
comprising nucleic acid(s) encoding heavy and/or light chain
sequences of antibodies or antigen-binding fragments of the
invention.
[0056] The production, characterization, and use of antibodies,
antigen-binding fragments, or other molecules specifically binding
hIL20 and having some or all of these properties are described in
more detail in the following sections.
Anti-IL20 Antibodies
[0057] The antibodies of the invention are characterized by
particular functional and/or structural features or properties of
the antibodies. Assays to evaluate the functional activities of
anti-IL20 antibodies are described in detail in separate sections
and in the Examples, and structural properties such as, e.g., amino
acid sequences, are described below.
[0058] Functional Properties
[0059] The antibodies of the invention bind specifically to hIL20.
The antibody preferably binds to hIL20 with high affinity, for
example with a KD of 10.sup.-7 M or less, a KD of 10.sup.-8 M or
less, a KD of 1 nM or less, a KD of about 0.3 nM or less, or a KD
of about 0.2 nM or less, or a KD of about 0.1 nM or less. A
recombinantly produced anti-IL20 antibody in sodium acetate buffer
may, for example, bind to recombinant hIL20 with an affinity of
about 0.1 nM or less, optionally with an affinity of about
0.01-0.05 nM, in a Biacore assay (see, e.g., Example 12).
Additionally, the antibodies may detectably bind to IL20 from one
or more non-human mammals, including mouse (e.g, mus musculus)
and/or cynomolgus monkey (Macaca fascicularis) (see, e.g., Example
2). Furthermore, the antibodies of the invention are capable of
reducing IL20-mediated activation of IL20R1/IL20R2 and
IL22R1/IL20R2 receptor complexes in vitro and/or in vivo. This may
be tested in one or more assays described herein (see, e.g.,
Examples 1, 2, and 9-11) or known in the art. Using a suitable
assay, an antibody of the invention can reduce hIL20-mediated
activation of human IL20R1/IL20R2 and IL22R1/IL20R2 receptor
complexes by at least about 10%, more preferably by at least 20%,
even more preferably by at least 30%, at least 40%, at least 50%,
or at least 60%, as compared to a control (e.g., in the absence of
any anti-hIL20 antibody). The antibody may further be able to
reduce IL20-mediated activation of IL20R1/IL20R2 and IL22R1/IL20R2
receptor complexes in other species, such as mice and cynomolgous
monkeys, using the corresponding ligand and receptor complex
orthologues (see Example 9).
[0060] The antibodies of the invention reduce IL20-mediated
proliferation of BaF-3 cells recombinantly expressing IL20R1/IL20R2
and IL22R1/IL20R2, but typically have no significant effect on
IL19- and/or IL24-induced proliferation (see, e.g., Example 2). In
such assays, an antibody of the invention typically reduces
proliferation with an EC50 of about 50 .mu.M or less, about 5 .mu.M
or less, about 1 .mu.M or less, about 0.5 .mu.M or less, about 0.1
.mu.M or less, about 0.05 .mu.M or less, or about 0.02 .mu.M or
less. For example, in a proliferation assay described in Example
10, recombinantly produced human antibody 15D2 had an EC50 of less
than 0.02 .mu.M.
[0061] The anti-IL20 antibodies of the invention can inhibit
hIL20-mediated receptor complex activation by any mechanism, or by
a combination of different mechanisms. Typically, an anti-hIL20
antibody can reduce or prevent hIL20 binding to cell-associated
hIL20 receptors or fully formed receptor complexes. Additionally or
alternatively, antibodies of the invention may bind to
cell-associated hIL20 single-chain receptor molecules, but prevent
formation of the receptor complex. Additionally or alternatively,
antibodies of the invention may bind to cell-associated hIL20
single-chain receptor molecules and fully formed receptor
complexes, but reduce or inhibit structural changes necessary for
receptor complex activation. Which one or more mechanisms are
involved can be identified by, e.g., testing whether the antibody
associates to cells expressing human IL20R1, IL20R2, IL22R1
receptor molecules, or IL20R1/IL20R2 and/or IL22R1/IL20R2 receptor
complexes in the presence of hIL20. In a specific embodiment, the
antibody reduces the binding of hIL20 to hIL20R2. In another
specific embodiment, the antibody does not reduce binding of hIL20
to at least one of the human IL20R1/IL20R2 and IL22R1/IL20R2
receptor complexes. Particular antibodies of the invention bind a
hIL20 epitope that at least partially overlaps, or includes at
least one residue in, the segment corresponding to Helix E in IL19,
optionally excluding D78. Without being limited to theory, this
segment can comprise or be part of a helical structure in IL20 that
is involved in binding to and/or activating IL20R1/IL20R2 and
IL22R1/IL20R2. For a model of hIL20 built using a hIL20-hIL19
sequence alignment and structural IL19 information, see FIG. 2. In
the hIL20 sequence, this segment comprises residues D78-H103 of
mature hIL20 (SEQ ID NO:1). In one embodiment, the antibody or
antigen-binding fragment of the invention thus binds to an epitope
comprising at least one residue selected from D78-H103 of mature
hIL20 (SEQ ID NO:1). In other specific and separate embodiments,
the epitope includes 2, 3, 4, 5, 6, 7 or more residues in the
D78-H103 segment.
[0062] In another aspect, the invention provides an antibody
binding an epitope comprising 1, 2, 3, 4, 5, 6, 7 or more residues
in the segment corresponding to residues D78-K96 or H79-K96 in
mature hIL20. This segment contains an epitope providing a higher
affinity of anti-IL20 antibody 5B7. The antibody may alternatively
bind an epitope comprising 1, 2, 3, 4, 5, 6, 7 or more residues in
the segment corresponding to residues D78-L93 or H79-L93, which
contains the key residues of the 15D2 epitope. For example, the
antibody may bind an epitope comprising at least one residue
selected from H79-L93. The antibody may alternatively bind an
epitope comprising 1, 2, 3, 4, 5, 6, 7 or more residues in the
segment corresponding to residues H79-N90, which contains the key
residues of the 5B7 epitope. In specific and separate embodiments,
all key residues of the epitope is in a segment corresponding to
residues D78-H103, D78-K96, D78-L93, or D78-N90, optionally
excluding D78.
[0063] In another aspect, the antibody binds an epitope comprising
at least one of residues H79, R83, S85, N90, F91, and L92 of mature
IL20. In separate and specific embodiments, the antibody binds 2,
3, 4, 5, 6, or all of D78, H79, R83, S85, N90, F91, and L92. In
another embodiment, the epitope comprises at least residues H79 and
N90. In an additional embodiment, the epitope further comprises
residue R83. In yet another embodiment, the epitope further
comprises 1, 2, 3, or all of D78, S85, F91, and L92 In another
aspect, the invention provides antibodies that compete with and/or
bind to the same epitope on hIL20 as an antibody comprising the VH
and VL sequences of either of 5B7 or 15D2, described below. Such
antibodies thus compete in binding to hIL20 with an antibody
comprising a light-chain variable region comprising SEQ ID NO:9 and
a heavy-chain variable region comprising SEQ ID NO:6 or SEQ ID
NO:7. Such antibodies can be identified based on their ability to
compete with 15D2 and/or 5B7 in standard hIL20 binding assays as
described herein (see, e.g., Example 4 or the section entitled
"Binding Assays" below). The ability of a test antibody to reduce
or inhibit the binding of 15D2 and/or 5B7 to hIL20 demonstrates
that the test antibody can compete with 15D2 and/or 5B7 for binding
to hIL20 and thus can bind to the same hIL20 segment or epitope as
5B7 and/or 15D2. In a preferred embodiment, the antibody that binds
to the same segment or epitope of hIL20 as 5B7 and/or 15D2 is a
human monoclonal antibody. Such human monoclonal antibodies can be
prepared and isolated according to known methods in the art, as
described herein.
[0064] In a particular embodiment, the antibody binds to a
different hIL20 segment or epitope than those bound by any of the
rat antibodies described in WO2005052000 (262.4.1.2.2.1,
262.5.1.6.4.4, and 262.7.1.3.2.4), and/or by murine antibodies (7E)
described in US20060142550 and WO2007081465, and competes more with
15D2 and/or 5B7 in binding to hIL20 than with either of the listed
mouse or rat antibodies. In another particular embodiment, the
antibody is a human antibody which does not bind to the segment
corresponding to residues 42-102 of the IL20 precursor (SEQ ID
NO:2).
[0065] Any combination of the above-described functional features,
other functional features described in the Examples, and/or
structural features describing in the following section, may be
exhibited by an antibody of the invention.
[0066] Structural Properties
[0067] In one aspect, the invention provides human anti-IL20
antibodies with suitable stability and/or solubility
characteristics for being formulated in aqueous formulations at
concentrations of at least about 50 mg/ml, at least about 60 mg/ml,
at least about 70 mg/ml, at least about 80 mg/ml, at least about 90
mg/ml, or at least about 100 mg/ml, which aqueous formulation may
further comprise a pharmaceutically acceptable excipient, diluent,
or carrier, and typically has a pH near neutral or physiological
pH. In one embodiment, the anti-IL20 antibody has a solubility of
at least 80 mg/ml in an aqueous formulation, optionally comprising
a 20 mM sodium phosphate buffer and 150 mM NaCl, and having a pH of
about 7.4. In one embodiment, the anti-IL20 antibody has a
solubility of at least 100 mg/ml in an aqueous formulation,
optionally comprising a 20 mM sodium phosphate buffer and 150 mM
NaCl, and having a pH of about 7.4. It has now been found that
human anti-IL20 antibodies deriving from certain germline sequences
are more soluble than others, thereby achieving higher
concentrations in an aqueous solution (see, e.g., Example 3). Such
embodiments are described in further detail below.
[0068] Preferred antibodies of the invention include the human
monoclonal antibodies 15D2 or 5B7 characterized as described
herein. Heavy and light chain variable domains and CDR sequences of
these antibodies are provided below and in FIG. 3,
[0069] The heavy chain variable domain of 15D2 (SEQ ID NO:6)
contains the following CDRs, corresponding to Kabat residues 31-35
(CDR1), 50-65 (CDR2) and 95-102 (CDR3) of SEQ ID NO:6,
respectively:
TABLE-US-00001 VH CDR1: NDIIH VH CDR2: WINAGYGNTQYSQNFQD VH CDR3:
EPLWFGESSPHDYYGMDV
[0070] The heavy chain variable domain of 5B7 (SEQ ID NO:7)
contains the following CDRs, corresponding to Kabat residues 31-35
(CDR1), 50-65 (CDR2) and 95-102 (CDR3) of SEQ ID NO:7,
respectively:
TABLE-US-00002 VH CDR1: SHIMH VH CDR2: WINAGYGNTKYSQNFQD VH CDR3:
EPLWFGELSPHDYYGMDV
[0071] The light chain variable domains of 15D2 and 5B7 (SEQ ID
NO:9) contains the following CDRs, corresponding to Kabat residues
24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) of SEQ ID NO:9,
respectively:
TABLE-US-00003 VL CDR1: RASQGISSALA VL CDR2: DASSLES VL CDR3:
QQFNSYPLT
[0072] Given that 15D2 and 5B7 both bind IL20, the VH CDR sequences
can be "mixed and matched" to create other anti-hIL20 binding
molecules of the invention. The hIL20-binding of such "mixed and
matched" antibodies can be tested using the binding assays
described herein (e.g. flow cytometry, Biacore, ELISAs) and/or
using a receptor-activation assay as described herein. The
invention thus provides antibodies that comprise the heavy chain
and light chain CDR1s, CDR2s and/or CDR3s of 15D2 or 5B7, or
combinations thereof. The CDR regions are delineated using the
Kabat system (FIG. 3). Given that each of these antibodies can bind
to hIL20 with substantially overlapping epitopes, and that
antigen-binding specificity is provided primarily by the CDR1, 2
and 3 regions, the VH CDR1, 2 and 3 sequences can be "mixed and
matched" (i.e., VH CDRs from different antibodies can be mixed and
matched, although each antibody can contain a VH CDR1, 2 and 3 and
a VL CDR1, 2 and 3) to create other anti-hIL20 binding molecules of
the invention. The 15D2 and 5B7 VH CDRs share substantial
structural similarity and are therefore amenable to mixing and
matching.
[0073] Accordingly, in one aspect, the invention provides an
isolated monoclonal antibody comprising: (a) a VH CDR1 from 5B7 or
15D2, (b) a VH CDR2 from 5B7 or 15D2, and (c) a VH CDR3 from 5B7 or
15D2, optionally combined with a VL sequence comprising the VL CDRs
of SEQ ID NO:9. This can also be illustrated using consensus VH
CDRs, per below.
[0074] The consensus variable heavy domain of 5B7115D2 contains the
following CDRs, corresponding to Kabat residues 31-35 (CDR1), 50-65
(CDR2) and 95-102 (CDR3) of SEQ ID NO:8, respectively, with X
representing any amino acid, preferably those listed below or
conservative substitutions thereof):
[0075] VH CDR1: X.sub.2X.sub.3IX.sub.4H (X.sub.2: N or S; X.sub.3:
D or H; X.sub.4: I or M, or conservative substitutions of any
thereof)
[0076] VH CDR2: WINAGYGNTX.sub.5YSQNFQD (X.sub.5 is K, Q, or a
conservative substitution of any thereof)
[0077] VH CDR3: EPLWFGEX.sub.7SPHDYYGMDV (X.sub.7 is S, L, or a
conservative substitution of any thereof),
[0078] wherein X.sub.2-X.sub.5 and X.sub.7 correspond to residues
31, 32, 34, 59, and 106 in SEQ ID NO:8, respectively.
[0079] Accordingly, in another aspect, the invention provides an
antibody comprising the heavy-chain variable regions CDR2 and CDR3,
optionally combined with the CDR1, of SEQ ID NO:8. In one
embodiment, the antibody comprises the sequence of SEQ ID NO:8. In
one aspect, the antibody comprises the heavy-chain variable region
CDR2 and CDR3, optionally combined with the CDR1, of SEQ ID NO:6.
In one embodiment, the antibody comprises the sequence of SEQ ID
NO:6. In one aspect, the antibody comprises the heavy-chain
variable region CDR2 and CDR3, optionally combined with the CDR1,
of SEQ ID NO:7. In one embodiment, the antibody comprises the
sequence of SEQ ID NO:7. In any of these aspects or embodiments,
the antibody may optionally further comprise the light-chain
variable regions CDR1, CDR2 and CDR3, or the full sequence, of SEQ
ID NO:9.
[0080] In certain embodiments, an antibody of the invention
comprises a VH region from a particular germline H chain
immunoglobulin gene, or a combination of particular germline H
chain immunoglobulin genes; and/or a VL region from a particular
germline L chain immunoglobulin gene, or a combination of
particular germline L chain immunoglobulin genes.
[0081] For example, in one embodiment, the invention provides an
isolated anti-hIL20 antibody comprising a heavy chain variable
region that is derived from a set of human genes comprising
VH1.sub.--03, D3-10, and JH6 genes. The heavy chain variable region
may, for example, comprise the CDR2 and CDR3 sequences, and
optionally the CDR1 sequence, of SEQ ID NO:8, respectively
corresponding to Kabat residues 50-65, 95-102, and 31-35. In
another embodiment, the antibody further comprises a light chain
variable region that is derived from a set of human genes
comprising VKI_L18 and JK4 genes. The light-chain variable region
may, for example, comprise the CDR1-CDR3 sequences of SEQ ID
NO:9.
[0082] In one embodiment, the invention provides an isolated
anti-hIL20 monoclonal antibody, or an antigen-binding fragment
thereof, wherein the antibody: (a) comprises a VH domain derived
from a human VH1.sub.--03 gene recombined with a human D3-10 gene
and a JH6 gene, (b) comprises a VL domain derived from a human
VKI_L18 gene recombined with a human JK4 gene, and (c) the antibody
specifically binds to hIL20. For example, the antibody may comprise
the light chain variable sequence of SEQ ID NO:9 and the
heavy-chain variable sequence of SEQ ID NO:6 or SEQ ID NO:7 As used
herein, a human antibody comprises heavy or light chain variable
regions of or "derived from" or "the product of" a particular
germline sequence if the variable regions of the antibody are
obtained from a system that uses human germline immunoglobulin
genes. Such systems include immunizing a transgenic mouse carrying
human immunoglobulin genes with the antigen of interest or
screening a human immunoglobulin gene library displayed on phage
with the antigen of interest. A human antibody that is "of" or
"derived from" or "the product of" a human germline immunoglobulin
sequence can be identified as such by comparing the amino acid
sequence of the human antibody to the amino acid sequences of human
germline immunoglobulins and selecting the human germline
immunoglobulin sequence that is closest in sequence (i.e., greatest
% identity) to the sequence of the human antibody. A human antibody
that is "of" or "derived from" or "the product of" a particular
human germline immunoglobulin sequence may contain amino acid
differences as compared to the germline sequence, due to, for
example, naturally-occurring somatic mutations or intentional
introduction of site-directed mutation.
[0083] However, a selected human antibody typically is at least 90%
identical in amino acid sequence to an amino acid sequence encoded
by a human germline immunoglobulin gene and contains amino acid
residues that identify the human antibody as being human when
compared to the germline immunoglobulin amino acid sequences of
other species (e.g., murine germline sequences). In certain cases,
a human antibody variable sequence may be at least 95%, or even at
least 96%, 97%, 98%, or 99% identical in amino acid sequence to the
amino acid sequence encoded by the recombined germline
immunoglobulin gene.
[0084] Typically, a human antibody derived from a particular human
germline sequence will display no more than 10 amino acid
differences from the amino acid sequence encoded by the human
germline immunoglobulin gene. In certain cases, the human antibody
may display no more than 8, no more than 5, or even no more than 4,
3, 2, or 1 amino acid difference, or no amino acid difference, from
the amino acid sequence encoded by the recombined germline
immunoglobulin gene.
[0085] In yet another aspect, an antibody of the invention
comprises heavy and light chain variable regions comprising amino
acid sequences that are homologous or identical to the amino acid
sequences of the preferred 15D2 and 5B7 antibodies described
herein, and wherein the antibodies retain the desired functional
properties of the anti-hIL20 antibodies of the invention. For
example, the invention provides an isolated monoclonal antibody
comprising a heavy chain variable domain and a light chain variable
domain, wherein: (a) the VH domain comprises an amino acid sequence
that is at least 80% identical to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 6, 7, and 8; (b) the VL
region comprises an amino acid sequence that is at least 80%
identical to SEQ ID NO:9; and (c) the antibody specifically binds
to hIL20 and exhibits at least one of the functional properties
described herein, preferably several of the functional properties
described herein.
[0086] In other embodiments, the VH and/or VL amino acid sequences
may be 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
sequences set forth above. An antibody having VH and VL regions
having high (i.e., 80% or greater) identity to the VH and VL
regions of the sequences set forth above, can be obtained by
mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of
nucleic acid molecules encoding SEQ ID NOs:6-9, followed by testing
of the encoded altered antibody for retained function (e.g., hIL20
binding affinity or reduction of hIL20-mediated activation of its
receptor complexes) using the functional assays described
herein.
[0087] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences (i.e.,
% identity=# of identical positions/total # of
positions.times.100), taking into account the number of gaps, and
the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm in sequence analysis
software. Protein analysis software matches similar sequences using
measures of similarity assigned to various substitutions, deletions
and other modifications, including conservative amino acid
substitutions.
[0088] The percent identity between two amino acid sequences can be
determined, e.g., using the Needleman and Wunsch (J. Mol. Biol.
48:444-453 (1970)) algorithm which has been incorporated into the
GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.
[0089] Polypeptide sequences can also be compared using FASTA,
applying default or recommended parameters. A program in GCG
Version 6.1., FASTA (e.g., FASTA2 and FASTA3) provides alignments
and percent sequence identity of the regions of the best overlap
between the query and search sequences (Pearson, Methods Enzymol.
1990; 183:63-98; Pearson, Methods Mol. Biol. 2000;
132:185-219).
[0090] The sequence identity between two amino acid sequences can
also be determined using the algorithm of E. Meyers and W. Miller
(Comput. Appl. Biosci., 1988; 11-17) which has been incorporated
into the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[0091] Another algorithm for comparing a sequence to another
sequence contained in a database is the computer program BLAST,
especially blastp, using default parameters. See, e.g., Altschul et
al., J. Mol. Biol. 1990; 215:403-410; Altschul et al., Nucleic
Acids Res. 1997; 25:3389-402 (1997); each herein incorporated by
reference. The protein sequences of the present invention can there
be used as a "query sequence" to perform a search against public
databases to, for example, identify related sequences. Such
searches can be performed using the XBLAST program (version 2.0) of
Altschul, et al. 1990 (supra). BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain
amino acid sequences homologous to the antibody molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al., 1997
(supra). When utilizing BLAST and Gapped BLAST programs, default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www. ncbi.nlm.nih.gov.
[0092] In certain embodiments, an antibody of the invention
comprises a VH region comprising CDR1, CDR2 and CDR3 sequences and
a VL region comprising CDR1, CDR2 and CDR3 sequences, wherein one
or more of these CDR sequences comprise specified amino acid
sequences based on the preferred antibodies described herein; 15D2
and 5B7, or conservative modifications thereof, and wherein the
antibodies retain the desired functional properties of the
anti-hIL20 antibodies of the invention. Accordingly, the invention
provides an isolated monoclonal antibody, or antigen-binding
fragment thereof, comprising a heavy chain variable region
comprising CDR1, CDR2, and CDR3 sequences and a light chain
variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
(a) the VH region CDR3 sequence comprises an amino acid sequence
selected from the group consisting of the CDR3 of SEQ ID NOs:6 and
7, and conservative modifications thereof; (b) the VL region CDR3
sequence comprises the amino acid sequence of the CDR3 of SEQ ID
NO:9 or conservative modifications thereof; and (c) the antibody
specifically binds to hIL20 and exhibits at least one of the
functional properties described herein, more preferably several of
the functional properties described herein.
[0093] In a further embodiment, the VH region CDR2 sequence
comprises an amino acid sequence selected from the group consisting
of the CDR2 of SEQ ID NOS: 6 or 7, and conservative modifications
thereof; and the VL region CDR2 sequence comprises the CDR2 of SEQ
ID NO:9 or conservative modifications thereof.
[0094] In a further embodiment, the VH region CDR1 sequence
comprises an amino acid sequence selected from the group consisting
of the CDR1 of SEQ ID NOS: 6 or 7, and conservative modifications
thereof; and the VL region CDR1 sequence comprises the CDR1 of SEQ
ID NO:9 or conservative modifications thereof.
[0095] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
[0096] "Conservative" amino acid substitutions are typically those
in which an amino acid residue is replaced with an amino acid
residue having a side chain with similar physicochemical
properties. Families of amino acid residues having similar side
chains have been defined in the art. These families include amino
acids with basic side chains (e.g., lysine, arginine, histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g. glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine, tryptophan), nonpolar side chains
(e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine), beta-branched side chains (e.g.
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine).
[0097] Thus, one or more amino acid residues within the CDR regions
of an antibody of the invention can be replaced with other amino
acid residues from the same side chain family and the altered
antibody can be tested for retained function (i.e., the functions
set forth in (c), (d) and (e) above) using the functional assays
described herein.
[0098] Antigen-Binding Fragments
[0099] The anti-hIL20 antibodies of the invention as described
herein may be prepared as full-length antibodies or antigen-binding
fragments thereof. Full-length antibodies can be of any suitable
class including, e.g., IgG and IgM. The specific class and/or
isotype of an antibody can be chosen according to the intended
therapeutic use. For example, the IgG1, IgG2, IgG3, and IgG4
isotypes have different affinities for Fc-receptors expressed on,
e.g., leukocytes, with IgG4 and IgG2 having lower affinities than
IgG1 and IgG3.
[0100] Examples of antigen-binding fragments include Fab, Fab',
F(ab)2, F(ab')2, F(ab)3, Fv (typically the VL and VH domains of a
single arm of an antibody), single-chain Fv (scFv; see e.g., Bird
et al., Science 1988; 242:423-426; and Huston et al. PNAS 1988;
85:5879-5883), dsFv, Fd (typically the VH and CH1 domain), and dAb
(typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains;
monovalent molecules comprising a single VH and a single VL chain;
minibodies, diabodies, triabodies, tetrabodies, and kappa bodies
(see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camel IgG;
IgNAR; as well as one or more isolated CDRs or a functional
paratope, where the isolated CDRs or antigen-binding residues or
polypeptides can be associated or linked together so as to form a
functional antibody fragment. Various types of antibody fragments
have been described or reviewed in, e.g., Holliger and Hudson, Nat
Biotechnol 2005; 23:1126-1136; WO2005040219, and published U.S.
Patent Applications 20050238646 and 20020161201.
[0101] Antibody fragments can be obtained using conventional
recombinant or protein engineering techniques, and the fragments
can be screened for antigen-binding or other function in the same
manner as are intact antibodies.
[0102] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of full-length antibodies (see, e.g.,
Morimoto et al., Journal of Biochemical and Biophysical Methods,
24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)).
However, these fragments can now be produced directly by
recombinant host cells. Alternatively, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab'):2 fragments (Carter et al., Bio/Technology, 10:163-167
(1992)). According to another approach, F(ab')2 fragments can be
isolated directly from recombinant host cell culture. In other
embodiments, the antibody of choice is a single-chain Fv fragment
(scFv). See WO 1993/16185; U.S. Pat. No. 5,571,894; and U.S. Pat.
No. 5,587,458. The antibody fragment may also be a "linear
antibody", e.g., as described in U.S. Pat. No. 5,641,870, for
example. Such linear antibody fragments may be monospecific or
bispecific.
[0103] Multispecific Molecules
[0104] In another aspect, the present invention features
multispecific molecules comprising an anti-hIL20 antibody, or an
antigen-fragment thereof, of the invention. Such multispecific
molecules include bispecific molecules comprising at least one
first binding specificity for hIL20 and a second binding
specificity for a second target epitope.
[0105] One type of bispecific molecules are bispecific antibodies.
Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Methods for
making bispecific antibodies are known in the art, and traditional
production of full-length bispecific antibodies is usually based on
the coexpression of two immunoglobulin heavy-chain-light-chain
pairs, where the two chains have different specificities (Millstein
et al., Nature, 305: 537-539 (1983)). Bispecific antibodies can be
prepared as full-length antibodies or antibody fragments (e.g.
F(ab')2 bispecific antibodies) or any other antigen-binding
fragments described herein.
[0106] Other multispecific molecules include those produced from
the fusion of a hIL20-binding antibody moiety to one or more other
non-antibody proteins. Such multispecific proteins and how to
construct them have been described in the art. See, e.g., Dreier et
al. (Bioconjug. Chem. 9(4): 482-489 (1998)); U.S. Pat. No.
6,046,310; U.S. Patent Publication No. 20030103984; European Patent
Application 1 413 316; US Patent Publication No. 20040038339; von
Strandmann et al., Blood (2006; 107:1955-1962.), and WO
2004056873.
[0107] Multispecific molecules with more than two valencies are
also contemplated. For example, trispecific antibodies can be
prepared. Tutt et al., J. Immunol, 147: 60 (1991).
[0108] The multispecific molecules of the present invention can be
prepared by conjugating the constituent binding specificities using
methods known in the art. For example, each binding specificity of
the multispecific molecule can be generated separately and then
conjugated to one another. When the binding specificities are
proteins or peptides, a variety of coupling or cross-linking agents
can be used for covalent conjugation. Examples of cross-linking
agents include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus (1985)
Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science
229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0109] When the binding specificities are antibodies, they can be
conjugated via sulthydryl bonding of the C-terminus hinge regions
of the two heavy chains. In a particularly preferred embodiment,
the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
[0110] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab')2 or
ligand.times.Fab fusion protein. A bispecific molecule of the
invention can be a single chain molecule comprising one single
chain antibody and a binding determinant, or a single chain
bispecific molecule comprising two binding determinants. Bispecific
molecules may comprise at least two single chain molecules. Methods
for preparing bispecific molecules are described or reviewed in,
for example in U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030;
U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. No.
5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S.
Pat. No. 5,258,498; U.S. Pat. No. 5,482,858; U.S. Patent
application publication 20030078385, Kontermann et al., (2005) Acta
Pharmacological Sinica 26(1):1-9; Kostelny et al., (1992) J.
Immunol. 148(5):1547-1553; Hollinger et al., (1993) PNAS (USA)
90:6444-6448; and Gruber et al. (1994) J. Immunol. 152: 5368.
Antibody Variants
[0111] An antibody of the invention further can be prepared using
an antibody having one or more of the VH and/or VL sequences
disclosed herein as starting material to engineer a modified
antibody or antibody "variant", which modified antibody may have
altered properties from the parent antibody. An antibody can be
engineered by modifying one or more residues within one or both
variable regions (i.e., VH and/or VL), for example within one or
more CDR regions and/or within one or more framework regions.
Additionally or alternatively, an antibody can be engineered by
modifying residues within the constant region(s), for example to
alter the effector function(s) of the antibody. Additionally, from
antigen-binding portions of an antibody, other constructs such as
antigen-binding fragments, antibody derivatives, immunoconjugates,
and multispecific molecules can be prepared.
[0112] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence.
[0113] Though an antibody variant or derivative typically has at
least one altered property as compared to the "parent" antibody,
the antibody variant or derivative can retain one, some or most of
the functional properties of the anti-hIL20 antibodies described
herein, which functional properties include, but are not limited
to: (a) reduces hIL20-mediated activation of human IL20R1/IL20R2
and IL22R1/IL20R2 receptor complexes, (b) binds to murine and
cynomolgous IL20 orthologs, preferably with substantially similar
efficacy or affinity; (c) competes with one or more of 15D2 and 5B7
in binding to hIL20, and (d) binds to an epitope in the segment
corresponding to Helix E (FIG. 2). Any combination of the
above-described functional features, and/or the functional features
as described in the Examples, may be exhibited by an antibody of
the invention.
[0114] The functional properties of the antibody variants and
derivatives can be assessed using standard assays available in the
art and/or described herein. For example, the ability of the
antibody to bind hIL20 can be determined using standard binding
assays, such as those set forth in the Examples (e.g., Biacore,
flow cytometry, or ELISAs).
[0115] Variable Region Modifications
[0116] One type of variable region engineering that can be
performed is CDR grafting. Antibodies interact with target antigens
predominantly through amino acid residues that are located in the
six heavy and light chain complementarily determining regions
(CDRs). For this reason, the amino acid sequences within CDRs are
more diverse between individual antibodies than sequences outside
of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g., Riechmann, L. et al. (1998)
Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525;
Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A.
86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.)
Accordingly, another embodiment of the invention pertains to an
isolated monoclonal antibody, or antigen binding portion thereof,
comprising the VH and/or VL CDR sequences of monoclonal antibodies
15D2 or 5B7, but framework sequences different from these
antibodies.
[0117] Framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "dBase"
human germline sequence database (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The
Repertoire of Human Germline VH Sequences Reveals about Fifty
Groups of VH Segments with Different Hypervariable Loops" J. Mol.
Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A Directory of
Human Germ-line VH Segments Reveals a Strong Bias in their Usage"
Eur. J. Immunol. 24:827-836; the contents of each of which are
expressly incorporated herein by reference.
[0118] Preferred framework sequences for use in the antibodies of
the invention are those that are structurally similar to the
framework sequences used by selected antibodies of the invention,
e.g., similar to the VH1.sub.--03, D3-10, JH6, VKI_L18, and/or JK4
sequences of 15D2 or 5B7. The VH CDR1, 2 and 3 sequences of 15D2 or
5B7 can be grafted onto framework regions that have the same
sequence as that found in the germline immunoglobulin gene from
which the framework sequence derive, or the CDR sequences can be
grafted onto framework regions that contain one or more mutations
as compared to the germline sequences. For example, it has been
found that in certain instances it is beneficial to mutate residues
within the framework regions to maintain or enhance the antigen
binding ability of the antibody (see e.g., U.S. Pat. Nos.
5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
[0119] In another aspect of the invention, the structural features
of anti-hIL20 antibodies of the invention, e.g., 15D2 and 5B7, are
used to create structurally related anti-hIL20 antibodies that
retain at least one functional property of the antibodies of the
invention, such as binding to hIL20. For example, one or more CDR
regions of 5B7 or 15D2, or variants thereof, can be combined
recombinantly with known framework regions and/or other CDRs to
create additional, recombinantly-engineered, anti-hIL20 antibodies
of the invention. The starting material for the engineering method
is one or more of the VH and/or VL sequences provided herein, or
one or more CDR regions thereof. To create the engineered antibody,
it is not necessary to actually prepare (i.e., express as a
protein) an antibody having one or more of the VH and/or VL
sequences provided herein, or one or more CDR regions thereof.
Rather, the information contained in the sequence(s) is used as the
starting material to create a "second generation" sequence(s)
derived from the original sequence(s) and then the "second
generation" sequence(s) is prepared and expressed as a protein.
[0120] Accordingly, in another embodiment, the invention provides a
method for preparing an anti-hIL20 antibody comprising: (a)
providing: (i) a heavy chain variable region antibody sequence
comprising CDR1, CDR2, and CDR3 sequences from SEQ ID NOS:6 or 7,
and (ii) a light chain variable region antibody sequence comprising
CDR sequences from SEQ ID NO:9; (b) altering at least one amino
acid residue within the first antibody sequence and/or the second
antibody sequence to create at least one altered antibody sequence;
and (c) preparing the altered antibody sequence; and (d) expressing
the altered antibody sequence as a protein.
[0121] Another type of variable region modification is to mutate
amino acid residues within the VH and/or VL CDR1, CDR2 and/or CDR3
regions to thereby improve one or more binding properties (e.g.,
affinity) of the antibody of interest. Site-directed mutagenesis or
PCR-mediated mutagenesis can be performed to introduce the
mutation(s) and the effect on antibody binding, or other functional
property of interest, can be evaluated in in vitro or in vivo
assays as described herein and provided in the Examples. Preferably
conservative modifications (as discussed above) are introduced. The
mutations may be amino acid substitutions, additions or deletions.
Moreover, typically no more than 8, more typically no more than 5
residues are altered within a single CDR region.
[0122] Accordingly, in another embodiment, the invention provides
isolated anti-hIL20 monoclonal antibodies, or antigen-binding
fragments thereof, comprising a heavy chain variable region
comprising VH CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6 or 7,
or amino acid sequences having one, two, three, four, five, six,
seven, or eight amino acid substitutions, deletions or additions in
one or more of these CDRs; and a light chain variable region
comprising VL CDR1, CDR2, and CDR3 sequences from SEQ ID NO:9, or
amino acid sequences having one, two, three, four, five, six,
seven, or eight amino acid substitutions, deletions or additions in
one or more of these CDRs.
[0123] Engineered antibodies of the invention include those in
which modifications have been made to framework residues within VH
and/or VL, e.g. to improve the properties of the antibody.
Typically such framework modifications are made to decrease the
immunogenicity of the antibody. For example, one approach is to
"backmutate" one or more framework residues to the corresponding
germline sequence. More specifically, an antibody that has
undergone somatic mutation may contain framework residues that
differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived.
[0124] For example, for 15D2 and 5B7, the VH residues that are
different from the corresponding germline sequence are indicated by
"*" in FIG. 3. To return, e.g., the framework region sequences to
their germline configuration, the somatic mutations outside the
CDRs can be "backmutated" to the germline sequence by, for example,
site-directed mutagenesis or PCR-mediated mutagenesis (e.g.,
residues 13, 68, and/or 82A of the 15D2 VH domain or residues 13,
30 and/or 82A of the 5B7 VH domain can be "backmutated" from the VH
amino acid to the germline amino acid. Such "backmutated"
antibodies are also intended to be encompassed by the
invention.
[0125] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 20030153043 by Carr et al.
[0126] Fc Modifications
[0127] In addition or as an alternative to modifications made
within the framework or CDR regions, antibodies of the invention
may be engineered to include modifications within the Fc region,
typically to alter one or more functional properties of the
antibody, such as serum half-life, complement fixation, Fc receptor
binding, protein stability and/or antigen-dependent cellular
cytotoxicity, or lack thereof. Furthermore, an antibody of the
invention may be chemically modified (e.g., one or more chemical
moieties can be attached to the antibody) or be modified to alter
its glycosylation, again to alter one or more functional properties
of the antibody. Each of these embodiments is described in further
detail below. The residues in the Fc region are numbered according
to Kabat.
[0128] If desired, the class of an antibody may be "switched" by
known techniques. Such techniques include, e.g., the use of direct
recombinant techniques (see e.g., U.S. Pat. No. 4,816,397) and
cell-cell fusion techniques (see e.g., U.S. Pat. No. 5,916,771).
For example, an antibody that was originally produced as an IgM
molecule may be class switched to an IgG antibody. Class switching
techniques also may be used to convert one IgG subclass to another,
e.g., from IgG1 to IgG2. Thus, the effector function of the
antibodies of the invention may be changed by isotype switching to,
e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for
various therapeutic uses. Exemplary cDNA sequences for constant
regions are available via, e.g., GenBank, each of which
incorporated by reference in its entirety, are as follows:
[0129] Human IgG1 constant heavy chain region: GenBank accession
No.: J00228;
[0130] Human IgG2 constant heavy chain region: GenBank accession
No.: J00230;
[0131] Human IgG3 constant heavy chain region: GenBank accession
No.: X04646;
[0132] Human IgG4 constant heavy chain region: GenBank accession
No.: K01316; and
[0133] Human kappa light chain constant region: GenBank accession
No.: J00241.
[0134] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody.
[0135] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al. In another embodiment, the antibody is modified to
increase its biological half life. Various approaches are possible.
For example, one or more of the following mutations can be
introduced: T252L, T254S, T256F, as described in U.S. Pat. No.
6,277,375 to Ward. Alternatively, to increase the biological half
life, the antibody can be altered within the CH1 or CL region to
contain a salvage receptor binding epitope taken from two loops of
a CH2 domain of an Fc region of an IgG, as described in U.S. Pat.
Nos. 5,869,046 and 6,121,022 by Presta et al. In yet other
embodiments, the Fc region is altered by replacing at least one
amino acid residue with a different amino acid residue to alter the
effecter function(s) of the antibody. For example, one or more
amino acids selected from amino acid residues 234, 235, 236, 237,
297, 318, 320 and 322 can be replaced with a different amino acid
residue such that the antibody has an altered affinity for an
effector ligand but retains the antigen-binding ability of the
parent antibody. The effector ligand to which affinity is altered
can be, for example, an Fc receptor or the C1 component of
complement. This approach is described in further detail in U.S.
Pat. Nos. 5,624,821 and 5,648,260, both to Winter et al. In another
example, one or more amino acids selected from amino acid residues
329, 331 and 322 can be replaced with a different amino acid
residue such that the antibody has altered C1q binding and/or
reduced or abolished complement dependent cytotoxicity (CDC). This
approach is described in further detail in U.S. Pat. No. 6,194,551
by Idusogie et al. In another example, one or more amino acid
residues within amino acid positions 231 and 239 are altered to
thereby alter the ability of the antibody to fix complement. This
approach is described further in PCT Publication WO 94/29351 by
Bodmer et al. In yet another example, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fcy receptor by modifying one or more amino acids
at the following positions: 238, 239, 248, 249, 252, 254, 255, 256,
258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,
289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,
312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335,
337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416,
419, 430, 434, 435, 437, 438 or 439. This approach is described
further in PCT Publication WO 00/42072 by Presta. Moreover, the
binding sites on human IgG1 for FcyRI, FcyRII, FcyRIII and FcRn
have been mapped and variants with improved binding have been
described (see Shields, R. L. et al. (2001) J. Biol. Chem.
276:6591-6604). Specific mutations at positions 256, 290, 298, 333,
334 and 339 were shown to improve binding to FcRIII. Additionally,
the following combination mutants were shown to improve FcyRIII
binding: T256A/S298A, S298A/E333A, S298A/K224A and
S298A/E333A/K334A.
[0136] The constant region may further be modified to stabilize the
antibody, e.g., to reduce the risk of a bivalent antibody
separating into two monovalent VH-VL fragments. For example, in an
IgG4 constant region, the Serine (S, Ser) residue at position 241
according to the Kabat numbering system may be mutated to a proline
(P, Pro) residue to allow complete disulphide bridge formation at
the hinge (see, e.g., Angal et al., Mol. Immunol. 1993; 30:105-8).
According to the EU index numbering system, Kabat residue 241,
corresponds to residue 228.
[0137] Glycosylation Modifications
[0138] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycoslated antibody can be
made (i.e., the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
antigen. Such carbohydrate modifications can be accomplished by,
for example, altering one or more sites of glycosylation within the
antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co
et al. Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
the invention to thereby produce an antibody with altered
glycosylation. For example, EP1176195 by Hanai et al. describes a
cell line with a functionally disrupted FUT8 gene, which encodes a
fucosyl transferase, such that antibodies expressed in such a cell
line exhibit hypofucosylation. PCT Publication WO 03/035835 by
Presta describes a variant CHO cell line, Lecl3 cells, with reduced
ability to attach fucose to Asn(297)-linked carbohydrates, also
resulting in hypofucosylation of antibodies expressed in that host
cell (see also Shields, R. L. et al. (2002) J. Biol. Chem.
277:26733-26740). PCT Publication WO 99/54342 by Umana et al.
describes cell lines engineered to express glycoprotein-modifying
glycosyl transferases (e.g.,
beta(I,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 7:176 180).
[0139] In certain embodiments of the methods of engineering
antibodies of the invention, mutations can be introduced randomly
or selectively along all or part of an anti-hIL20 antibody coding
sequence (e.g., a 15D2 or 5B7 coding sequence) and the resulting
modified antibodies can be screened for binding activity and/or
other functional properties as described herein. Mutational methods
have been described in the art. For example, PCT Publication WO
02/092780 by Short describes methods for creating and screening
antibody mutations using saturation mutagenesis, synthetic ligation
assembly, or a combination thereof.
[0140] Alternatively, PCT Publication WO 03/074679 by Lazar et al.
describes methods of using computational screening methods to
optimize physiochemical properties of antibodies.
Antibody Derivatives
[0141] Antibody derivatives (or immunoconjugates) within the scope
of this invention include anti-hIL20 antibodies conjugated or
covalently bound to a second agent.
[0142] For example, in one aspect, the invention provides
immunoconjugates comprising an antibody conjugated or covalently
bonded to a cytotoxic agent. The term "cytotoxic agent" as used
herein is a molecule that is capable of killing a cell to which it
becomes associated, e.g., via IL20-binding to cell-surface hIL20
receptors. Any type of moiety with a cytotoxic or cytoinhibitory
effect can be conjugated to the present antibodies to form a
cytotoxic conjugate of the present invention, including therapeutic
radioisotopes, toxic proteins, toxic small molecules, such as
drugs, toxins, immunomodulators, hormones, hormone antagonists,
enzymes, oligonucleotides, enzyme inhibitors, therapeutic
radionuclides, angiogenesis inhibitors, chemotherapeutic drugs,
vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,
antimetabolites, alkylating agents, antibiotics, COX-2 inhibitors,
SN-38, antimitotics, antiangiogenic and apoptotoic agents,
particularly doxorubicin, methotrexate, taxol, CPT-11,
camptothecans, nitrogen mustards, gemcitabine, alkyl sulfonates,
nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs,
purine analogs, platinum coordination complexes, Pseudomonas
exotoxin, ricin, abrin, 5-fluorouridine, ribonuclease (RNase),
DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,
gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin and others (see, e.g., Remington's Pharmaceutical
Sciences, 19th Ed. (Mack Publishing Co. 1995); Goodman and Gilman's
The Pharmacological Basis of Therapeutics (McGraw Hill, 2001);
Pastan et al. (1986) Cell 47:641; Goldenberg (1994) Cancer Journal
for Clinicians 44:43; U.S. Pat. No. 6,077,499; the entire
disclosures of which are herein incorporated by reference). It will
be appreciated that a toxin can be of animal, plant, fungal, or
microbial origin, or can be created de novo by chemical
synthesis.
[0143] In another embodiment, the antibody is derivatized with a
radioactive isotope, such as a therapeutic radionuclide or a
radionuclide suitable for detection purposes. Any of a number of
suitable radioactive isotopes can be used, including, but not
limited to, I-131, Indium-111, Lutetium-171, Bismuth-212,
Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67,
Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33,
Scandium-47, Silver-111, Gallium-67, Praseodymium-142,
Samarium-153, Terbium-161, Dysprosium-166, Holmium-166,
Rhenium-186, Rhenium-188, Rhenium-189, Lead-212, Radium-223,
Actinium-225, Iron-59, Selenium-75, Arsenic-77, Strontium-89,
Molybdenum-99, Rhodium-105, Palladium-109, Praseodymium-143,
Promethium-149, Erbium-169, Iridium-194, Gold-198, Gold-199, and
Lead-211. In general, the radionuclide preferably has a decay
energy in the range of 20 to 6,000 keV, preferably in the ranges 60
to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,
and 4,000-6,000 keV for an alpha emitter. Also preferred are
radionuclides that substantially decay with generation of
alpha-particles.
[0144] The antibody conjugates of the invention can be used to
modify a given biological response, where the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL1"), interleukin-2 ("IL2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
or other growth factors.
[0145] The second agent can be linked to the antibody directly or
indirectly, using any of a large number of available methods. For
example, an agent can be attached at the hinge region of the
reduced antibody component via disulfide bond formation, using
cross-linkers such as N-succinyl 3-(2-pyridyldithio)proprionate
(SPDP), or via a carbohydrate moiety in the Fc region of the
antibody (see, e.g., Yu et al. (1994) Int. J. Cancer 56: 244; Wong,
Chemistry of Protein Conjugation and Cross-linking (CRC Press
1991); Upeslacis et al., "Modification of Antibodies by Chemical
Methods," in Monoclonal antibodies: principles and applications,
Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price,
"Production and Characterization of Synthetic Peptide-Derived
Antibodies," in Monoclonal antibodies: Production, engineering and
clinical application, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995), Cattel et al. (1989) Chemistry today
7:51-58, Delprino et al. (1993) J. Pharm. Sci 82:699-704; Arpicco
et al. (1997) Bioconjugate Chemistry 8:3; Reisfeld et al. (1989)
Antihody, Immunicon. Radiopharm. 2:217, the entire disclosure of
each of which is herein incorporated by reference). See, also, e.g.
Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In
Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985);
Hellstrom et al., "Antibodies For Drug Delivery", in Controlled
Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel
Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents
In Cancer Therapy: A Review", in Monoclonal Antibodies '84:
Biological And Clinical Applications, Pinchera et al. (eds.), pp.
475-506 (1985); "Analysis, Results, And Future Prospective Of The
Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in
Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et
al., (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al.,
"The Preparation And Cytotoxic Properties Of Antibody-Toxin
Conjugates", Immunol. Rev., 62:119-58 (1982).
[0146] For further discussion of types of cytotoxins, linkers and
methods for conjugating therapeutic agents to antibodies, see also
Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P.
A. et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G.
(2003) Cancer Cell 3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer
2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin.
Investig. Drugs 3:1089-1091; Senter, P. D. and Springer, C. J.
(2001) Adv. Drug Deliv. Rev. 53:247-264.
[0147] In other embodiments, the second agent is a detectable
moiety, which can be any molecule that can be quantitatively or
qualitatively observed or measured. Examples of detectable markers
useful in the conjugated antibodies of this invention are
radioisotopes, fluorescent dyes, or a member of a complementary
binding pair, such as a member of any one of: and antigen/antibody
(other than an antibody to IL20), lectin/carbohydrate;
avidin/biotin; receptor/ligand; or molecularly imprinted
polymer/print molecule systems.
[0148] The second agent may also or alternatively be a polymer,
intended to, e.g., increase the circulating half-life of the
antibody or antigen-binding fragment thereof. Exemplary polymers
and methods to attach such polymers to peptides are illustrated in,
e.g., U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and
4,609,546. Additional illustrative polymers include
polyoxyethylated polyols and polyethylene glycol (PEG) moieties. As
used herein, the term "polyethylene glycol" is intended to
encompass any of the forms of PEG that have been used to derivatize
other proteins, such as mono (C1-C10) alkoxy- or
aryloxy-polyethylene glycol or polyethylene glycol-maleimide. For
example, a full-length antibody or antibody fragment can be
conjugated to one or more PEG molecules with a molecular weight of
between about 1,000 and about 40,000, such as between about 2000
and about 20,000, e.g., about 3,000-12,000. To pegylate an antibody
or fragment thereof, the antibody or fragment typically is reacted
with polyethylene glycol (PEG), such as a reactive ester or
aldehyde derivative of PEG, under conditions in which one or more
PEG groups become attached to the antibody or antibody fragment.
Preferably, the pegylation is carried out via an acylation reaction
or an alkylation reaction with a reactive PEG molecule (or an
analogous reactive water-soluble polymer). In certain embodiments,
the antibody to be pegylated is an a glycosylated antibody. Methods
for pegylating proteins are known in the art and can be applied to
the antibodies of the invention. See for example, WO2004099231,
WO2003031464, EP154316 (by Nishimura et al.) and EP401384 (by
Ishikawa et al.).
Nucleic Acids
[0149] Another aspect of the invention pertains to nucleic acid
molecules that encode the antibodies of the invention. The nucleic
acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form. A nucleic acid is
"isolated" or "rendered substantially pure" when purified away from
other cellular components or other contaminants, e.g., other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols
in Molecular Biology, Greene Publishing and Wiley Interscience, New
York. A nucleic acid of the invention can be, for example, DNA or
RNA and may or may not contain intronic sequences. In a preferred
embodiment, the nucleic acid is a cDNA molecule.
[0150] Nucleic acids of the invention can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g., using phage display techniques), nucleic acids
encoding the antibody can be recovered from the library. Preferred
nucleic acids molecules of the invention are those encoding heavy
and light chain sequences of 15D2 or 5B7 monoclonal antibodies of
the IgG2 or IgG4 isotype, more preferably IgG4.
[0151] Once DNA fragments encoding VH and VL segments are obtained,
these DNA fragments can be further manipulated by standard
recombinant DNA techniques, for example to convert the variable
region genes to full-length antibody chain genes, to Fab fragment
genes or to a scFv gene. In these manipulations, a VL- or
VH-encoding DNA fragment is operatively linked to another DNA
fragment encoding another protein, such as an antibody constant
region or a flexible linker. The term "operatively linked", as used
in this context, is intended to mean that the two DNA fragments are
joined such that the amino acid sequences encoded by the two DNA
fragments remain in-frame.
[0152] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al. (I 991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG4 constant region. For a Fab fragment heavy
chain gene, the VH-encoding DNA can be operatively linked to
another DNA molecule encoding only the heavy chain CH1 constant
region.
[0153] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but most preferably is a kappa constant
region.
[0154] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such
that the VH and VL sequences can be expressed as a contiguous
single-chain protein, with the VL and VH regions joined by the
flexible linker (see e.g., Bird et al. (1988) Science 242:423-426;
Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883;
McCafferty et al., (1990) Nature 348:552-554).
Antibody Production
[0155] Monoclonal antibodies (mAbs) of the present invention can be
produced by a variety of techniques, including conventional
monoclonal antibody methodology e.g., the standard somatic cell
hybridization technique of Kohler and Milstein (1975) Nature 256:
495. Although somatic cell hybridization procedures are preferred,
in principle, other techniques for producing monoclonal antibody
can be employed e.g., viral or oncogenic transformation of B
lymphocytes.
[0156] One preferred animal system for preparing hybridomas is the
murine system. Immunization protocols and techniques for isolation
of immunized splenocytes for fusion are known in the art, as are
fusion partners (e.g., murine myeloma cells) and fusion procedures.
Chimeric or humanized antibodies of the present invention can also
be prepared based on the sequence of a murine monoclonal antibody
using established techniques. For example, DNA encoding the heavy
and light chain immunoglobulins can be obtained from the murine
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, the murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, the murine CDR
regions can be inserted into a human framework using methods known
in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.
Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0157] In a preferred embodiment, the antibodies of the invention
are human monoclonal antibodies. Such human monoclonal antibodies
directed against hIL20 can be generated using transgenic or
transchromosomic mice carrying parts of the human immune system
rather than the mouse system. These transgenic and transchromosomic
mice include mice referred to herein as HuMAb mice and KM mice,
respectively, and are collectively referred to herein as "human Ig
mice." The HuMAb mouse (Medarex, Inc.) contains human
immunoglobulin gene miniloci that encode unrearranged human heavy
(p and y) and K light chain immunoglobulin sequences, together with
targeted mutations that inactivate the endogenous, u and K chain
loci (see e.g., Lonberg, et al. (1994) Nature 368: 856-859).
Accordingly, the mice exhibit reduced expression of mouse IgM or K,
and, in response to immunization, the introduced human heavy and
light chain transgenes undergo class switching and somatic mutation
to generate high affinity human IgGK monoclonal (Lonberg, N. et al.
(1994), supra; reviewed in Lonberg, N. (1994) Handbook of
Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D.
(1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and
Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The
preparation and use of HuMab mice, and the genomic modifications
carried by such mice, is further described in Taylor, L. et al.
(1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993)
International Immunology 5: 647-656; Tuaillon et al. (1993) Proc.
Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature
Genetics 4: 117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;
Tuaillon et al. (1994) J. Immunol. 152:2912 2920; Taylor, L. et al.
(1994) International immunology 6: 579-591; and Fishwild, D. et al.
(1996) Nature Biotechnology 14: 845-851, the contents of all of
which are hereby specifically incorporated by reference in their
entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;
5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. No.
5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO
93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962,
all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to
Korman et al. In another embodiment, human antibodies of the
invention can be raised using a mouse that carries human
immunoglobulin sequences on transgenes and transchomosomes, such as
a mouse that carries a human heavy chain transgene and a human
light chain transchromosome. Such mice, referred to herein as "KM
mice", are described in detail in PCT Publication WO 02/43478 to
Ishida et al. Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-hIL20 antibodies of the invention. For
example, an alternative transgenic system referred to as the
Xenomouse (Abgenix, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598;
6,150,584 and 6,162,963 to Kucherlapati et al. Moreover,
alternative transchromosomic animal systems expressing human
immunoglobulin genes are available in the art and can be used to
raise anti-hIL20 antibodies of the invention. For example, mice
carrying both a human heavy chain transchromosome and a human light
chain tranchromosome, referred to as "TC mice" can be used; such
mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci.
USA 97:722-727. Furthermore, cows carrying human heavy and light
chain transchromosomes have been described in the art (Kuroiwa et
al. (2002) Nature Biotechnology 20:889-894) and can be used to
raise anti-hIL20 antibodies of the invention.
[0158] Human monoclonal antibodies of the invention can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art. See for
example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to
Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et
al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.;
and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;
6,582,915 and 6,593,081 to Griffiths et al. Human monoclonal
antibodies of the invention can also be prepared using SCID mice
into which human immune cells have been reconstituted such that a
human antibody response can be generated upon immunization. Such
mice are described in, for example, U.S. Pat. Nos. 5,476,996 and
5,698,767 to Wilson et al.
[0159] When human Ig mice are used to raise human antibodies of the
invention, such mice can be immunized with a purified or enriched
preparation of hIL20 antigen, as described by Lonberg, N. et al.
(1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996)
NatureBiotechnology 14: 845-851; and PCT Publication WO 98/24884
and WO 01/14424. Preferably, the mice will be 6-16 weeks of age
upon the first infusion. For example, a purified or enriched
preparation (5-50 .mu.g) of hIL20 antigen can be used to immunize
the human Ig mice intraperitoneally.
[0160] The form and amount of antigen administered (e.g., hIL20
polypeptide), as well as administration schedules and the possible
use of adjuvants such as, e.g., complete Freund's adjuvant or
incomplete Freund's adjuvant, are typically optimized for each
antigen-mouse system according to established methods in the
art.
[0161] The immune response can be monitored over the course of the
immunization protocol with plasma samples being obtained by
retroorbital bleeds, and the plasma or serum can be screened by
ELISA (as described below), and mice with sufficient titers of
anti-hIL20 human immunoglobulin can be used for fusions. Mice can
be boosted intravenously with antigen 3 days before sacrifice and
removal of the spleen. It is expected that 2-3 fusions for each
immunization may need to be performed.
[0162] To generate hybridomas producing human monoclonal antibodies
of the invention, splenocytes and/or lymph node cells from
immunized mice can be isolated and fused to an appropriate
immortalized cell line, such as a mouse myeloma cell line. The
resulting hybridomas can be screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice can be fused to
one-sixth the number of P3.times.63-Ag8.653 nonsecreting mouse
myeloma cells (ATCC, CRL 1580) with 50% PEG. Alternatively, the
cells can be fused by electrofusion. Cells are plated at
approximately 2.times.10.sup.5 in a flat bottom microtiter plate,
followed by a two week incubation in selective medium containing
20% fetal Clone Serum, 18% "653" conditioned media, 5% origen
(IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055
mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml
streptomycin, 50 mg/ml gentamycin and 1.times.HAT (Sigma; the HAT
is added 24 hours after the fusion). After approximately two weeks,
cells can be cultured in medium in which the HAT is replaced with
HT. Individual wells can then be screened by ELISA for human
monoclonal IgM and IgG antibodies. Once extensive hybridoma growth
occurs, medium can be observed usually after 10-14 days. The
antibody secreting hybridomas can be replated, screened again, and
if still positive for human IgG, the monoclonal antibodies can be
subcloned at least twice by limiting dilution. The stable subclones
can then be cultured in vitro to generate small amounts of antibody
in tissue culture medium for characterization. To purify human
monoclonal antibodies, selected hybridomas can be grown in
two-liter spinner-flasks for monoclonal antibody purification.
Supernatants can be filtered and concentrated before affinity
chromatography with protein A-sepharose (Pharmacia, Piscataway,
N.J.). Eluted IgG can be checked by gel electrophoresis and high
performance liquid chromatography to ensure purity. The buffer
solution can be exchanged into PBS, and the concentration can be
determined by spectroscopy. The monoclonal antibodies can be
aliquoted and stored at -80.degree.
[0163] Antibodies of the invention can also be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (e.g., Morrison, S. (1985) Science 229:1202).
[0164] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g. PCR amplification or cDNA cloning using a
hybridoma that expresses the antibody of interest) and the DNAs can
be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene.
[0165] The expression vector and expression control sequences are
chosen to be compatible with the expression host cell used. The
antibody light chain gene and the antibody heavy chain gene can be
inserted into separate vector or, more typically, both genes are
inserted into the same expression vector. The antibody genes are
inserted into the expression vector by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene
fragment and vector, or blunt end ligation if no restriction sites
are present). The light and heavy chain variable regions of the
antibodies described herein can be used to create full-length
antibody genes of any antibody isotype by inserting them into
expression vectors already encoding heavy chain constant and light
chain constant regions of the desired isotype such that the VH
segment is operatively linked to the CH segment(s) within the
vector and the VL segment is operatively linked to the CL segment
within the vector. Additionally or alternatively, the recombinant
expression vector can encode a signal peptide that facilitates
secretion of the antibody chain from a host cell. The antibody
chain gene can be cloned into the vector such that the signal
peptide is linked in-frame to the amino terminus of the antibody
chain gene. The signal peptide can be an immunoglobulin signal
peptide or a heterologous signal peptide (i.e., a signal peptide
from a non-immunoglobulin protein).
[0166] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology,
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990)).
[0167] It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV), Simian Virus 40
(SV40), adenovirus, (e.g., the adenovirus major late promoter
(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences
may be used, such as the ubiquitin promoter or p-globin promoter.
Still further, regulatory elements composed of sequences from
different sources, such as the SRa promoter system, which contains
sequences from the SV40 early promoter and the long terminal repeat
of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988)
Mol. Cell. Biol. 8:466-472).
[0168] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g. origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g. U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0169] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6:12-13).
[0170] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Nail. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells,
COS cells and SP2 cells. In particular, for use with NSO myeloma
cells, another preferred expression system is the GS gene
expression system disclosed in WO 87/04462, WO 89/01036 and EP
338,841. When recombinant expression vectors encoding antibody
genes are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. Antibodies can be
recovered from the culture medium using standard protein
purification methods.
Antibody Characterization
[0171] After production or purification, or as part of a screening
or selection procedure, the functional characteristics of an
anti-hIL20 antibody of the invention can be investigated.
Functional properties of interest include, e.g., antibody binding
specificity for hIL20, antibody binding to hIL20 orthologs,
antibody competition with reference antibodies (such as, e.g., 5B7
and 15D2), the epitope to which the antibody binds, the affinity of
the antibody-antigen interaction, antagonistic properties of the
antibody, and solubility.
[0172] The following are brief descriptions of exemplary assays for
antibody characterization. Some are further described in other
sections and/or the Examples.
[0173] Binding Assays
[0174] The present invention provides for antibodies, and
antigen-binding fragments, variants, and immunoconjugates thereof,
that bind hIL20. Any of a wide variety of assays can be used to
assess binding of an antibody to hIL20. Protocols based upon
ELISAs, radioimmunoassays, Western blotting, BIACORE, and other
competition assays, inter alia, are suitable for use and are well
known in the art. Further, several binding assays, including
competition assays, are described in the Examples.
[0175] For example, simple binding assays can be used, in which a
test antibody is incubated in the presence of a target protein or
epitope (e.g., IL20 or a portion thereof), unbound antibodies are
washed off, and the presence of bound antibodies is assessed using,
e.g., radiolabels, physical methods such as mass spectrometry, or
direct or indirect fluorescent labels detected using, e.g.,
cytofluorometric analysis (e.g. FACScan). Such methods are well
known to those of skill in the art. Any amount of binding above the
amount seen with a control, non-specific antibody indicates that
the antibody binds specifically to the target.
[0176] In such assays, the ability of the test antibody to bind to
the target cell or protein can be compared with the ability of a
(negative) control protein, e.g. an antibody raised against a
structurally unrelated antigen, or a non-Ig peptide or protein, to
bind to the same target. Antibodies or fragments that bind to the
target cells or IL20 using any suitable assay with 25%, 50%, 100%,
200%, 1000%, or higher increased affinity relative to the control
protein, are said to "specifically bind to" or "specifically
interact with" the target, and are preferred for use in the
therapeutic methods described below. The ability of a test antibody
to affect the binding of a (positive) control antibody against
IL20, e.g. 15D2 or 5B7, may also be assessed.
[0177] In one aspect, the invention provides for anti-hIL20
antibodies sharing biological characteristics and/or substantial VH
and/or VL sequence identity with 15D2 or 5B7. One exemplary
biological characteristic is the binding to the 15D2 or 5B7
epitope, or the respective regions in the extracellular domain of
hIL20 to which the 15D2 or 5B7 antibodies bind. To screen for
antibodies that bind to the 15D2 or 5B7 epitope, a routine
cross-blocking assay, such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed.
[0178] In an exemplary cross-blocking or competition assay, 15D2 or
5B7 (control) antibody and a test antibody are admixed (or
pre-adsorbed) and applied to a sample containing IL20. In certain
embodiments, one would pre-mix the control antibodies with varying
amounts of the test antibody (e.g., 1:10 or 1:100) for a period of
time prior to applying to the IL20-containing sample. In other
embodiments, the control and varying amounts of test antibody can
simply be admixed during exposure to the antigen/target sample. As
long as one can distinguish bound from free antibodies (e.g., by
using separation or washing techniques to eliminate unbound
antibodies) and the control antibody from test antibody (e.g., by
using species- or isotype-specific secondary antibodies, by
specifically labeling the control antibody with a detectable label,
or by using physical methods such as mass spectrometry to
distinguish between different compounds) one will be able to
determine if the test antibody reduces the binding of the control
antibody to the antigen, indicating that the test antibody
recognizes substantially the same epitope as the control. In this
assay, the binding of the (labeled) control antibody in the
presence of a completely irrelevant antibody is the control high
value. The control low value is be obtained by incubating the
labeled (positive) control antibody with unlabeled control
antibody, where competition would occur and reduce binding of the
labeled antibody.
[0179] In a test assay, a significant reduction in labeled antibody
reactivity in the presence of a test antibody is indicative of a
test antibody that competes with, or substantially binds to the
same epitope, as the control antibody. Any test antibody or
compound that reduces the binding of the labeled control to the
antigen/target by at least 50% or more preferably 70%, at any ratio
of control:test antibody or compound between about 1:10 and about
1:100 is considered to be an antibody or compound that competes
with or binds to substantially the same epitope or determinant as
the control. Preferably, such test antibody or compound will reduce
the binding of the control to the antigen/target by at least 90%.
Nevertheless, any compound or antibody that reduces the binding of
a control antibody or compound to any measurable extent can be used
in the present invention.
[0180] In one embodiment, competition can be assessed by a flow
cytometry test. Cells bearing hIL20 are incubated first with a
control antibody that is known to specifically bind to
hIL20receptor, and then with the test antibody which may be
labelled with, e.g., a fluorochrome or biotin. The test antibody is
said to compete with the control if the binding obtained with
preincubation with saturating amounts of control antibody is 80%,
preferably, 50, 40 or less of the binding (mean of fluorescence)
obtained by the antibody without preincubation with the control.
Alternatively, a test antibody is said to compete with the control
if the binding obtained with a labeled control (by a fluorochrome
or biotin) on cells preincubated with saturating amount of antibody
to test is 80%, preferably 50%, 40%, or less of the binding
obtained without preincubation with the antibody. See Example 4 for
an exemplary antibody competition assay.
[0181] Functional Assays
[0182] The antibodies of the invention are capable of reducing
IL20-mediated activation of IL20R1/IL20R2 and IL22R1/IL20R2
receptor complexes in vitro and/or in vivo. Various suitable assays
are known in the art.
[0183] For example, the Examples describe a luciferase assay that
detects receptor complex activation based on the following
principles. Briefly, upon IL20 binding and receptor complex
formation, the associated JAK kinases are autophosphorylated and
can phosphorylate STAT3 protein. Phosphorylated STAT3 can enter the
nucleus and bind a specific DNA element of the promoter that has
been placed next to a gene encoding luciferase. Luciferase is then
expressed, and can transform the substrate luciferin to light,
which can then be detected and quantified.
[0184] Another type of in vitro assay for testing of IL20R1/IL20R2
and IL22R1/IL20R2 receptor activation is based on proliferation of,
e.g., BaF-3 cells transfected with IL20 receptor complexes from
humans or other species. Such an assay can test for a neutralizing
effect of IL20-induced proliferation of BaF-3 cells transfected
with, e.g., hIL20R1/hIL20R2 or hIL22R1/hIL20R2. The BaF-3 cells are
IL3 dependent, and proliferate in vitro after the addition of IL3
in their growth medium. If IL3 is not available, the cells will die
within a few days, showing signs of apoptosis already after a few
hours of IL3 starvation. When the transfected BaF-3 cells are
stimulated through their IL20 receptor complex, they will start to
divide and do not need IL3. Specific assays for inhibition of
proliferation are described in the Examples,
[0185] Solubility Assays
[0186] Suitable assays for testing the solubility, i.e., the
concentration of antibody that can be achieved in a solution, are
described in Example 3 and in Harris, E.L.V. (1989) In Harris,
E.L.V. and Angal, S. (eds), Protein Purification Methods. A
Practical Approach. IRL Press, New York, see, e.g., pp.
131-133.
Pharmaceutical Formulations
[0187] In one embodiment, the present invention provides
pharmaceutical composition comprising anti-hIL20 antibodies as
described herein together with one or more carriers. The human
antibodies of the invention, including 15D2 and 5B7, are suitable
for pharmaceutical formulations, where a high concentration is
often advantageous or necessary, e.g., when used for subcutaneous
administration.
[0188] The pharmaceutical formulations and administration modes
described in WO2006003179, hereby incorporated by reference in
their entireties, can also be used for the antibodies and
applications of the present invention.
[0189] One object of the invention is to provide a pharmaceutical
formulation comprising an antibody of the invention which is
present in a concentration from 1 mg/ml to 150 mg/ml, from 1 mg/ml
to 200 mg/ml, or from 1 mg/ml to 500 mg/ml, typically in an aqueous
or freeze-dried formulation (for reconstitution), and wherein said
formulation has a pH from 2.0 to 10.0, typically around neutral pH.
Preferably, in an aqueous formulation, the antibody is present in
soluble form at concentrations of least about 50 mg/ml, at least
about 60 mg/ml, at least about 70 mg/ml, at least about 80 mg/ml,
at least about 90 mg/ml, or at least about 100 mg/ml. The
formulation may further comprise one or more pharmaceutically
acceptable excipients, diluents, and/or carriers. These may
include, e.g., a buffer system, as well as one or more
preservatives, tonicity agents, chelating agents, stabilizers
and/or surfactants. Suitable carriers are known in the art and
described in, e.g., Remington: The Science and Practice of
Pharmacy, 19.sup.th edition, 1995.
[0190] Suitable antibody formulations can also be determined by
examining experiences with other already developed therapeutic
monoclonal antibodies. Several monoclonal antibodies have been
shown to be efficient in clinical situations, such as Rituxan
(Rituximab), Herceptin (Trastuzumab) Xolair (Omalizumab), Bexxar
(Tositumomab), Campath (Alemtuzumab), Zevalin, Oncolym, Humira and
similar formulations may be used with the antibodies of this
invention. For example, a monoclonal antibody can be supplied at a
concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50
mL) single-use vials, formulated for IV administration in 9.0 mg/mL
sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL
polysorbate 80, and sterile water for injection. The pH is adjusted
to 6.5. Alternatively, the antibody can be formulated in a solution
comprising a phosphate buffer, or histidine, sucrose, and
Polysorbate 80.
Diagnostic Applications
[0191] The hIL20-antibodies of the invention also have
non-therapeutic applications. For example, anti-hIL20 antibodies
may also be useful in diagnostic assays for IL20 protein, e.g.
detecting its presence in specific tissues, tissue samples (e.g.,
synovial fluid) or in serum.
[0192] For diagnostic applications, the antibody typically will be
labelled with a detectable moiety. Numerous labels are available
that can be generally grouped into the following categories:
[0193] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The antibody can be labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, Volumes 1 and 2, Coligen et al., Ed.
Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and
radioactivity can be measured using scintillation counting.
[0194] (b) Fluorescent labels such as rare-earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorimeter.
[0195] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally catalyzes a chemical alteration of the chromogenic
substrate that can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Examples of enzymatic labels include luciferases (e.g.,
firefly luciferase and bacterial luciferase; U.S. Pat. No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate
dehydrogenase, urease, peroxidase such as horseradish peroxidase
(HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase,
lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose
oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic
oxidases (such as uricase and xanthine oxidase), lactoperoxidase,
microperoxidase, and the like. Techniques for conjugating enzymes
to antibodies are described in O'Sullivan et al, "Methods for the
Preparation of Enzyme-Antibody Conjugates for use in Enzyme
Immunoassay," in Methods in Enzymology (Ed., J. Langone & H.
Van Vunakis), Academic Press, New York, 73:147-166 (1981).
[0196] The antibodies may also be used for in vivo diagnostic
assays. Generally, the antibody is labeled with a radionuclide or a
non-radioactive indicator detectable by, e.g., nuclear magnetic
resonance, or other means known in the art. Preferably, the label
is a radiolabel, such as, e.g., .sup.125I, .sup.131I, .sup.67Cu,
.sup.99mTc, or .sup.111In. The labelled antibody is administered to
a host, preferably via the bloodstream, and the presence and
location of the labelled antibody in the host is assayed. This
imaging technique is suitably used in the detection, staging, or
treatment of the diseases or disorders in question, for example,
rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis,
psoriatic arthritis, ankylosing spondylitis, Sjogren's syndrome,
multiple sclerosis, inflammatory bowel disease, systemic lupus
erythematosus and/or lupus nephritis, by visualisation of IL20 in
synovial fluids and -membranes. The radioisotope is conjugated to
the protein by any means, including metal-chelating compounds
iodogen techniques for iodination.
[0197] As a matter of convenience, the antibodies of the present
invention can be provided in a kit, i.e., a packaged combination of
reagents in predetermined amounts with instructions for performing
the diagnostic assay. Where the antibody is labelled with an
enzyme, the kit will include substrates and cofactors required by
the enzyme (e.g., a substrate precursor that provides the
detectable chromophore or fluorophore). In addition, other
additives may be included such as stabilizers, buffers (e.g., a
block buffer or lysis buffer) and the like. The relative amounts of
the various reagents may be varied widely to provide for
concentrations in solution of the reagents that substantially
optimize the sensitivity of the assay. Particularly, the reagents
may be provided as dry powders, usually lyophilized, including
excipients that on dissolution will provide a reagent solution
having the appropriate concentration.
Therapeutic Applications
[0198] Methods of treating a patient using a human or humanized
anti-hIL20 antibody as described herein are also provided for by
the present invention. In one embodiment, the invention provides
for the use of a human or humanized antibody as described herein in
the preparation of a pharmaceutical composition for administration
to a human patient. Typically, the patient suffers from, or is at
risk for, an autoimmune or inflammatory disease or disorder.
[0199] In one aspect, the conditions or disorders to be treated
with the antibodies and other compounds of the invention are
rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis,
psoriatic arthritis, ankylosing spondylitis, Sjogren's syndrome,
multiple sclerosis, inflammatory bowel diseases such as ulcerative
colitis and Crohn's disease, systemic lupus erythematosus, or lupus
nephritis, and any combination thereof, as well as co-morbidities
associated with these diseases, with cardiovascular disease being a
non-limiting example of said co-morbidities. In a further aspect,
other exemplary conditions include, but are not limited to,
juvenile chronic arthritis, osteoarthritis, other
spondyloarthropathies than ankylosing spondylitis, systemic
sclerosis (scleroderma), idiopathic inflammatory myopathies
(dermatomyositis, polymyositis), vasculitis, systemic vasculitis,
temporal arteritis, atherosclerosis, sarcoidosis, myasthenia
gravis, autoimmune hemolytic anemia (immune pancytopenia,
paroxysmal nocturnal hemoglobinuria), pernicious anemia, autoimmune
thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia), thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis), diabetes mellitus, Type 2 diabetes, immune-mediated
renal disease (glomerulonephritis, tubulointerstitial nephritis,
autoimmune oophiritis), pancreatitis, autoimmune orchitis,
autoimmune uveitis, anti-phospholipid syndrome, demyelinating
diseases of the central and peripheral nervous systems in addition
to multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy, hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B. C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, viral hepatitis, primary
biliary cirrhosis, granulomatous hepatitis, Wegener's
granulomatosis, Behcet's disease, and sclerosing cholangitis,
inflammatory bowel diseases such as celiac disease,
gluten-sensitive enteropathy, and Whipple's disease, autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, atopic dermatitis,
dermitis herpetiformis, pemphigus vulgaris, vitiligo (leukoderma),
allergic diseases such as asthma, allergic rhinitis, atopic
dermatitis, food hypersensitivity and urticaria, sepsis,
endotoxemia, immunologic diseases of the lung such as eosinophilic
pneumonias, idiopathic pulmonary fibrosis and hypersensitivity
pneumonitis, chronic obstructive pulmonary disease, and organ or
bone marrow transplantation associated diseases including graft
rejection and graft-versus-host-disease.
[0200] For example, in one aspect, the anti-IL20 antibody is used
in combination with one or more other anti-inflammatory agents,
including, but not limited to, analgesic agents, immunosuppressive
agents (e.g., B- or T-cell antagonists such as B-cell depletion
agents and T cell inhibiting agents; complement inhibiting agents),
corticosteroids, and anti-TNFalpha agents or other anti-cytokine or
anti-cytokine receptor agents, and anti-angiogenic agents. Specific
examples include metothrexate, TSG-6, Rituxan.RTM. or other B-cell
therapies, anti-IL12 antibodies, CTLA4-Fc fusion proteins,
IL1-receptor antagonists, IL1 antibodies, IL15 antibodies, IL18
antibodies, and anti-IL6R antibodies. Further examples of
combination therapies are provided below.
[0201] When one or more other agents or approaches are used in
combination with the present therapy, there is no requirement for
the combined results to be additive of the effects observed when
each treatment is conducted separately. Although at least additive
effects are generally desirable, any decrease in IL20 activity or
other beneficial effect above one of the single therapies would be
of benefit. Also, there is no particular requirement for the
combined treatment to exhibit synergistic effects, although this is
certainly possible and advantageous. The IL20-based treatment may
precede, or follow, the other treatment by, e.g., intervals ranging
from minutes to weeks and months. It also is envisioned that more
than one administration of either the anti-IL20 composition or the
other agent will be utilized. The agents may be administered
interchangeably, on alternate days or weeks; or a cycle of
anti-IL20 treatment may be given, followed by a cycle of the other
agent therapy. In any event, all that is required is to deliver
both agents in a combined amount effective to exert a
therapeutically beneficial effect, irrespective of the times for
administration.
[0202] Rheumatoid Arthritis
[0203] Rheumatoid arthritis (RA) is a chronic systemic autoimmune
inflammatory disease that mainly involves the synovial membrane of
multiple joints with resultant injury to the articular cartilage.
The pathogenesis is T lymphocyte dependent and is associated with
the production of rheumatoid factors, auto antibodies directed
against self IgG, with the resultant formation of immune complexes
that attain high levels in joint fluid and blood. These complexes
in the joint may induce the marked infiltrate of lymphocytes and
monocytes into the synovium and subsequent marked synovial changes;
the joint space is infiltrated by similar cells with the addition
of numerous neutrophils. Tissues affected are primarily the joints,
often in symmetrical pattern. However, extra-articular disease also
occurs in two major forms. One form is the development of
extra-articular lesions with ongoing progressive joint disease and
typical lesions of pulmonary fibrosis, vasculitis, and cutaneous
ulcers. The second form of extra-articular disease is the so called
Felty's syndrome which occurs late in the RA disease course,
sometimes after joint disease has become quiescent, and involves
the presence of neutropenia, thrombocytopenia and splenomegaly.
This can be accompanied by vasculitis in multiple organs with
formations of infarcts, skin ulcers and gangrene. Patients often
also develop rheumatoid nodules in the subcutis tissue overlying
affected joints; the nodules late stage have necrotic centers
surrounded by a mixed inflammatory cell infiltrate. Other
manifestations which can occur in RA include: pericarditis,
pleuritis, coronary arteritis, intestitial pneumonitis with
pulmonary fibrosis, keratoconjunctivitis sicca, and rheumatoid
nodules. IL20 has been demonstrated in rheumatoid arthritis
synovial fluid (Hsu et al. (2006) Arthritis Rheum. 54, 2722-2733;
Kragstrup et al., (2008) Cytokine 41, 16-23), and IL20 receptor
expression has been demonstrated in rheumatoid arthritis synovium
(Hsu et al., (2006) Arthritis Rheum. 54, 2722-2733; Sakurai et al.,
(2008) Rheumatology (Oxford) 47, 815-820).
[0204] Accordingly, in one aspect, the invention provides a method
for treating and/or preventing rheumatoid arthritis (RA). The
method comprises delivering an effective amount of an anti-hIL20
antibody to a patient having RA or being identified/diagnosed as
being at substantial risk of developing RA, such that RA is treated
or prevented. In a further aspect, the antibody that is capable of
detectably reducing IL20-mediated activation of IL20R1/IL20R2 and
IL22R1/IL20R2 receptor complexes. In one aspect, the method results
in a modulation of one or more biomarkers in a manner consistent
with the treatment or prevention (as applicable) of RA (e.g., serum
IL-6, TNF-.alpha., IL1, VEGF, TIFF R, IL2R, shed CD4, shed CD8,
and/or C reactive protein). In vivo models of RA in which the
antibodies of the invention can optionally be tested are described
in U.S. Pat. No. 6,414,218 and US Patent Publication No.
20030005469 (related principles and models are described in, e.g.,
Wooley, P. H., Animal Models of Arthritis, eds. J. H. Klippel and
P. A. Dieppe, Mosby Publishers (London), 1998; Erning et al.,
Arthritis Res, 4 Suppl 3:S 133-40, 2002; Holmdahl et al., Ageing
Res Rev, 1(1): 135-47, 2002; Anthony et al., Clin Exp Rheumatol,
17(2):240-4, 1999; Durie et al., Clin Immuno) Immunopathol,
73(1):11-8, 1994; and Muller-Ladner et al., Drugs Today (Bare),
35(4-5):379-88, 1999).
[0205] In another aspect, the practice of the method results in a
detectable reduction of synovial inflammation in the peripheral
joints of the patient/host. In one aspect, the method results in
preventing radiographic deterioration and improving physical
function in the patient or host as exhibited by, e.g., a reduction
in radiographic progression in the patient or host, reduction in
swollen and tender joints (as determined by acceptable analytical
criteria), and/or significantly improved quality of life (e.g., as
determined by a reduction in disability scores on the RA Health
Assessment Questionnaire). The antibody can be used alone or in
combination with one or more other anti-RA agent, such as a
non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, an
analgesic, a corticosteroid (e.g., predinisone, hydrocortisone),
gold, an immunosuppressant (e.g., methotrexate), a B-cell depletion
agent (e.g., Rituxan.RTM.), a B-cell agonist (e.g.,
LymphoStat-B.RTM.) and an anti-TNFalpha agent (e.g., Embrel.RTM.,
Humira.RTM. and Remicade.RTM.), an anti-IL1 receptor antagonist
(e.g., Kineret.RTM.), an anti-IL15 antibody, or a disease-modifying
anti-rheumatic drug (DMARD).
[0206] Demyelinating Diseases
[0207] Demyelinating diseases of the central and peripheral nervous
systems, including Multiple Sclerosis (MS); idiopathic
demyelinating polyneuropathy or Guillain-Barre syndrome; and
Chronic Inflammatory Demyelinating Polyneuropathy, are believed to
have an autoimmune basis and result in nerve demyelination as a
result of damage caused to oligodendrocytes or to myelin directly.
In MS there is evidence to suggest that disease induction and
progression is dependent on T lymphocytes. MS is a demyelinating
disease that is T lymphocyte-dependent and has either a
relapsing-remitting course or a chronic progressive course. The
etiology is unknown; however, viral infections, genetic
predisposition, environment, and autoimmunity all contribute.
Lesions contain infiltrates of predominantly T lymphocyte mediated,
microglial cells and infiltrating macrophages; CD4+ T lymphocytes
are the predominant cell type at lesions.
[0208] Thus, in another aspect, the invention provides a method for
treating and/or preventing MS. The method comprises delivering an
effective amount of an anti-hIL20 antibody to a human patient
having MS or being identified/diagnosed as being at substantial
risk of developing MS, such that MS is treated or prevented in the
patient or host. The antibody can be used alone or in combination
with other anti-MS agents such as Tyzabri.RTM..
[0209] Inflammatory Bowel Disease
[0210] In another aspect, the invention provides a method for
treating and/or preventing inflammatory bowel disease (IBD), such
as Crohn's disease or ulcerative colitis.
[0211] The method of treating an inflammatory bowel disease
comprises delivering an effective amount of an anti-IL20 antibody
to a human patient having IBD or being identified/diagnosed as
being at substantial risk of developing IBD, such that IBD is
treated or prevented in the patient. The antibody can be used alone
or in combination with other anti-IBD agents, such as drugs
containing mesalamine (including sulfasalazine and other agents
containing 5-aminosalicylic acid (5-ASA), such as olsalazine and
balsalazide), non-steroidal anti-inflammatory drugs (NSAIDs),
analgesics, corticosteroids (e.g., predinisone, hydrocortisone),
TNF-inhibitors (including adilimumab (Humira.RTM., etanercept
(Enbrel.RTM. and infliximab (Remicade.RTM.), anti-IL12 antibodies,
immunosuppressants (such as 6-mercaptopurine, azathioprine and
cyclosporine A), and antibiotics.
[0212] Psoriasis
[0213] Psoriasis is a T lymphocyte-mediated inflammatory disease.
Lesions contain infiltrates of T lymphocytes, macrophages and
antigen processing cells, and some neutrophils. IL20 and its
receptors are present in elevated levels in psoriatic lesions (Wei
et al., Clin Immuno) (2005) 117: 65-72; Romer et al., J Invest
Dermatol 2003; 121, 1306-1311; Wang et al. J Invest Dermatol 2006;
126: 1590-1599; Otkjr et al., Br J Dermatol 2005; 153:
911-918).
[0214] Thus, in another aspect, the invention provides a method for
treating and/or preventing psoriasis. The method comprises
delivering an effective amount of an anti-hIL20 antibody to a human
patient having psoriasis or being identified/diagnosed as being at
substantial risk of developing psoriasis, such that psoriasis is
treated or prevented in the patient. The antibody can be used alone
or in combination with one or more other anti-psoriasis treatments
such as phototherapy, topical therapy (e.g., tar, topical
glucocorticoids), or systemic therapy (e.g., methotrexate, a
synthetic retinoid, cyclosporine), an anti-TNFalpha agent (e.g.,
Embrel.RTM., Humira.RTM., Remicade.RTM.), a T-cell inhibitor (e.g.,
Raptiva.RTM.), vitamin D analogs, p38 mitogen-activated protein
kinase (MAPK) inhibitors, as well as a biologic agent such as
Rituxan.RTM..
[0215] Psoriatic Arthritis
[0216] Psoriatic arthritis is a chronic inflammatory arthritic
condition affecting the skin, the joints, the insertion sites of
tendons, ligaments, and fascia, and is commonly associated with
psoriasis. (Approximately 7% of patients with psoriasis develop
psoriatic arthritis). Much evidence suggests that a T-cell-mediated
process drives the pathophysiology of psoriatic arthritis.
Monocytes also play a role in psoriatic arthritis and are
responsible for the production of matrix metalloproteinases, which
may mediate the destructive changes in the joints of patients with
psoriatic arthritis.
[0217] Thus, in another aspect, the invention provides a method for
treating and/or preventing psoriatic arthritis. The method
comprises delivering an effective amount of an anti-hIL20 antibody
to a human patient having psoriatic arthritis or being
identified/diagnosed as being at substantial risk of developing
psoriatic arthritis, such that the psoriatic arthritis is treated
or prevented in the patient. The antibody can be used alone or in
combination with one or more other anti-psoriatic arthritis
treatments such as nonsteroidal anti-inflammatory drugs (aspirin,
ibuprofen), methotrexate, a synthetic retinoid, cyclosporine, a
corticosteroid, an anti-TNFalpha agent (e.g., Embrel.RTM.,
Humira.RTM., Remicade.RTM.).
[0218] Systemic Lupus Erythematosus
[0219] In systemic lupus erythematosus (SLE), the central mediator
of disease is the production of auto-reactive antibodies to self
proteins/tissues and the subsequent generation of immune-mediated
inflammation. Antibodies either directly or indirectly mediate
tissue injury. Though T lymphocytes have not been shown to be
directly involved in tissue damage, T lymphocytes are required for
the development of auto-reactive antibodies. The genesis of the
disease is thus T lymphocyte dependent. Multiple organs and systems
are affected clinically including kidney, lung, musculoskeletal
system, mucocutaneous, eye, central nervous system, cardiovascular
system, gastrointestinal tract, bone marrow and blood.
[0220] Thus, in another aspect, the invention provides a method for
treating and/or preventing SLE. The method comprises delivering an
effective amount of an anti-hIL20 antibody to a human patient
having SLE or being identified/diagnosed as being at substantial
risk of developing SLE, such that the SLE is treated or prevented
in the patient. The antibody can be used alone or in combination
with other anti-SLE agents, such as non-steroidal anti-inflammatory
drugs (NSAIDs), analgesics, corticosteroids (e.g., predinisone,
hydrocortisone), immunosuppressants (such as cyclophosphamide,
azathioprine, and methotrexate), antimalarials (such as
hydroxychloroquine) and biologic drugs that inhibit the production
of dsDNA antibodies (e.g. LIP 394).
[0221] Diabetes
[0222] Type I diabetes mellitus or insulin-dependent diabetes is
the autoimmune destruction of pancreatic islet B cells; this
destruction is mediated by auto-antibodies and auto-reactive T
cells. Antibodies to insulin or the insulin receptor can also
produce the phenotype of insulin-non-responsiveness.
[0223] Thus, in another aspect, an anti-IL20 antibody is delivered
to a patient suffering from or at substantial risk of developing
type I diabetes mellitus in an amount and under conditions
sufficient to treat or prevent the condition in the patient. The
antibody can be used alone or in combination with other
anti-diabetic agents, such as insulin, or beta cell growth or
survival factors, or immunomodulatory antibodies such as anti-CD3
antibodies.
[0224] Transplantation
[0225] Transplantation associated diseases, including graft
rejection and Graft-Versus-Host-Disease (GVHD), is T
lymphocyte-dependent; inhibition of T lymphocyte function is
ameliorative.
[0226] Thus, in another aspect, the invention provides methods of
reducing the likelihood of transplant rejection (or reducing the
severity or prolonging the time to onset of a transplant
rejection-related condition, i.e., to prolong allograft survival).
The method comprises delivering an effective amount of an
anti-hIL20 antibody to a human patient that is about to be, is, or
recently was the recipient of a tissue/organ transplant, such that
the likelihood of rejection is detectably reduced (e.g., as
compared to a control). Examples of tissue transplants that can be
treated include, but are not limited to, liver, lung, kidney,
heart, small bowel, and pancreatic islet cells, as well as in bone
marrow-transplantation and in the treatment of graft versus host
disease (GVHD). The antibody can be used alone or in combination
with other agents for inhibiting transplant rejection, such as
immunosuppressive agents (e.g. cyclosporine, azathioprine,
methylprednisolone, prednisolone, prednisone, mycophenolate
mofetil, sirilimus, rapamycin, tacrolimus), anti-infective agents
(e.g., acyclovir, clotrimazole, ganciclovir, nystatin,
trimethoprimsulfarnethoxazole), diuretics (e.g. bumetanide,
furosemide, metolazone) and ulcer medications (e.g., cimetidine,
farnotidine, lansoprazole, omeprazole, ranitidine, sucralfate). For
hematopoietic transplantation, hematopoietic growth factor(s)
(e.g., erythropoietin, G-CSF, GM-CSF, IL3, IL11, thrombopoietin,
etc.) or antimicrobial(s) (e.g., antibiotic, antiviral, antifungal)
may be administered as an adjunct therapy.
[0227] Other Autoimmune or Inflammatory Diseases
[0228] In other separate aspects, the invention provides methods
for treating and/or preventing other autoimmune or inflammatory
diseases or disorders, comprising delivering an effective amount of
an anti-hIL20 antibody to a human patient having the disease or
disorder or being identified/diagnosed as being at substantial risk
of developing the disease or disorder, such that it is treated or
prevented in the patient, where the disease or disorder is one
described below. The antibody can be used alone or in combination
with one or more other therapeutic agents used for treating the
disease or disorder.
[0229] Juvenile chronic arthritis is a chronic idiopathic
inflammatory disease which begins often at less than 16 years of
age. Its phenotype has some similarities to RA; some patients which
are rheumatoid factor positive are classified as juvenile
rheumatoid arthritis. The disease is sub-classified into three
major categories: pauciarticular, polyarticular, and systemic. The
arthritis can be severe and is typically destructive and leads to
joint ankylosis and retarded growth. Other manifestations can
include chronic anterior uveitis and systemic amyloidosis.
[0230] Spondyloarthropathies are a group of disorders with some
common clinical features and the common association with the
expression of HLA-B27 gene product. The disorders include:
ankylosing spondylitis, Reiter's syndrome (reactive arthritis),
arthritis associated with inflammatory bowel disease, spondylitis
associated with psoriasis, juvenile onset spondyloarthropathy and
undifferentiated spondyloarthropathy. Distinguishing features
include sacroileitis with or without spondylitis; inflammatory
asymmetric arthritis; association with HLA-B27 (a serologically
defined allele of the HLA-B locus of class I MHC); ocular
inflammation, and absence of autoantibodies associated with other
rheumatoid disease. The cell most implicated as key to induction of
the disease is the CD8+ T lymphocyte, a cell which targets antigen
presented by class I MHC molecules. CD8+ T cells may react against
the class I MHC allele HLA B27 as if it were a foreign peptide
expressed by MHC class I molecules. It has been hypothesized that
an epitope of HLA-B27 may mimic a bacterial or other microbial
antigenic epitope and thus induces a CD8+ T cells response.
[0231] Systemic sclerosis (scleroderma) has an unknown etiology. A
hallmark of the disease is induration of the skin; likely this is
induced by an active inflammatory process. Scleroderma can be
localized or systemic; vascular lesions are common and endothelial
cell injury in the microvasculature is an early and important event
in the development of systemic sclerosis; the vascular injury may
be immune mediated. An immunologic basis is implied by the presence
of mononuclear cell infiltrates in the cutaneous lesions and the
presence of anti-nuclear antibodies in many patients. ICAM-1 is
often unregulated on the cell surface of fibroblasts in skin
lesions suggesting that T cell interaction with these cells may
have a role in the pathogenesis of the disease. Other organs
involved include: the gastrointestinal tract: smooth muscle atrophy
and fibrosis resulting in abnormal peristalsis/motility; kidney:
concentric subendothelial intimal proliferation affecting small
arcuate and interlobular arteries with resultant reduced renal
cortical blood flow, results in proteinuria, azotemia and
hypertension; skeletal muscle: atrophy, interstitial fibrosis;
inflammation; lung: interstitial pneumonitis and interstitial
fibrosis; and heart: contraction band necrosis,
scarring/fibrosis.
[0232] Idiopathic inflammatory myopathies including
dermatomyositis, polymyositis and others are disorders of chronic
muscle inflammation of unknown etiology resulting in muscle
weakness. Muscle injury/inflammation is often symmetric and
progressive. Autoantibodies are associated with most forms. These
myositis-specific autoantibodies are directed against and inhibit
the function of components, proteins and RNA's, involved in protein
synthesis.
[0233] Sjogren's syndrome is due to immune-mediated inflammation
and subsequent functional destruction of the tear glands and
salivary glands. The disease can be associated with or accompanied
by inflammatory connective tissue diseases. The disease is
associated with autoantibody production against Ro and La antigens,
both of which are small RNA-protein complexes. Lesions result in
keratoconjunctivitis sicca, xerostomia, with other manifestations
or associations including bilary cirrhosis, peripheral or sensory
neuropathy, and palpable purpura.
[0234] Systemic vasculitis are diseases in which the primary lesion
is inflammation and subsequent damage to blood vessels which
results in ischemia/necrosis/degeneration to tissues supplied by
the affected vessels and eventual end-organ dysfunction in some
cases. Vasculitides can also occur as a secondary lesion or
sequelae to other immune-inflammatory mediated diseases such as
rheumatoid arthritis, systemic sclerosis, etc., particularly in
diseases also associated with the formation of immune complexes.
Diseases in the primary systemic vasculitis group include: systemic
necrotizing vasculitis: polyarteritis nodosa, allergic angiitis and
granulomatosis, polyangiitis; Wegener's granulomatosis;
lymphomatoid granulomatosis; and giant cell arteritis.
Miscellaneous vasculitides include: mucocutaneous lymph node
syndrome (MLNS or Kawasaki's disease), isolated CNS vasculitis,
Behet's disease, thromboangiitis obliterans (Buerger's disease) and
cutaneous necrotizing venulitis. The pathogenic mechanism of most
of the types of vasculitis listed is believed to be primarily due
to the deposition of immunoglobulin complexes in the vessel wall
and subsequent induction of an inflammatory response either via
ADCC, complement activation, or both.
[0235] Sarcoidosis is a condition of unknown etiology which is
characterized by the presence of epithelioid granulomas in nearly
any tissue in the body; involvement of the lung is most common. The
pathogenesis involves the persistence of activated macrophages and
lymphoid cells at sites of the disease with subsequent chronic
sequelae resultant from the release of locally and systemically
active products released by these cell types.
[0236] Autoimmune hemolytic anemia including autoimmune hemolytic
anemia, immune pancytopenia, and paroxysmal noctural hemoglobinuria
is a result of production of antibodies that react with antigens
expressed on the surface of red blood cells (and in some cases
other blood cells including platelets as well) and is a reflection
of the removal of those antibody coated cells via complement
mediated lysis and/or ADCC/Fc-receptor-mediated mechanisms.
[0237] In autoimmune thrombocytopenia including thrombocytopenic
purpura, and immune-mediated thrombocytopenia in other clinical
settings, platelet destruction/removal occurs as a result of either
antibody or complement attaching to platelets and subsequent
removal by complement lysis, ADCC or Fc-receptor mediated
mechanisms.
[0238] Thyroiditis including Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, and atrophic
thyroiditis, are the result of an autoimmune response against
thyroid antigens with production of antibodies that react with
proteins present in and often specific for the thyroid gland.
Experimental models exist including spontaneous models: rats (BUF
and BB rats) and chickens (obese chicken strain); inducible models:
immunization of animals with either thyroglobulin, thyroid
microsomal antigen (thyroid peroxidase).
[0239] Immune mediated renal diseases, including glomerulonephritis
and tubulointerstitial nephritis, are the result of antibody or T
lymphocyte mediated injury to renal tissue either directly as a
result of the production of autoreactive antibodies or T cells
against renal antigens or indirectly as a result of the deposition
of antibodies and/or immune complexes in the kidney that are
reactive against other, non-renal antigens. Thus other
immune-mediated diseases that result in the formation of
immune-complexes can also induce immune mediated renal disease as
an indirect sequelae. Both direct and indirect immune mechanisms
result in inflammatory response that produces/induces lesion
development in renal tissues with resultant organ function
impairment and in some cases progression to renal failure. Both
humoral and cellular immune mechanisms can be involved in the
pathogenesis of lesions.
[0240] Inflammatory and Fibrotic Lung Disease, including
Eosinophilic Pneumonias; Idiopathic Pulmonary Fibrosis, and
Hypersensitivity Pneumonitis may involve a disregulated
immune-inflammatory response. Inhibition of such a response would
be of therapeutic benefit.
[0241] Autoimmune or Immune-mediated Skin Disease including Bullous
Skin Diseases, Erythema Multiforme, and Contact Dermatitis are
mediated by auto-antibodies, the genesis of which is T lymphocyte
dependent.
[0242] Allergic diseases, including asthma; allergic rhinitis;
atopic dermatitis; food hypersensitivity; and urticaria are T
lymphocyte dependent. These diseases are predominantly mediated by
T lymphocyte induced inflammation, IgE mediated-inflammation or a
combination of both.
[0243] It will be understood that the effective amount of the IL20
antibody, as well as the overall dosage regimen, may vary according
to the disease and the patient's clinical status, which, in turn,
may be reflected in one or more clinical parameters such as
clinically accepted disease scores. For example, for rheumatoid
arthritis, the severity of disease and/or outcome of treatment may
be evaluated by monitoring number of swollen joints; pain;
mobility; and/or the official disease score ACR 20/50 or 70. For
Type 1 diabetes, severity of disease and/or outcome of treatment
may be evaluated by measuring blood glucose levels or variations
thereof, HbIC levels, the amount of insulin needed, and the like.
For multiple sclerosis, brain inflammation can be assessed through
scanning the brain. For hematopoietic transplant rejection,
severity of the disease (failure to engraft) and/or outcome of
treatment may be evaluated by evidence of prolonged neutropenia,
thrombocytopenia, and red-cell transfusion dependence in patients
that have undergone myeloablative conditioning, and by failure to
observe chimerism in patients that have undergone non-myeloablative
conditioning. In general, detectable effects on treatment outcome
using the methods and compositions of the present invention include
a decrease in the necessity for other treatments (including, e.g.,
a decrease in the amount and/or duration of other drugs or
treatments), a decrease in number and/or duration of hospital
stays, a decrease in lost work days due to illness, and the like.
It will be further understood that the effective amount may be
determined by those of ordinary skill in the art by routine
experimentation, by constructing a matrix of values and testing
different points in the matrix.
[0244] Dosages
[0245] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example, dosages can be about 0.3 mg/kg body
weight, about 1 mg/kg body weight, about 3 mg/kg body weight, about
5 mg/kg body weight or about 10 mg/kg body weight or within the
range of 1-10 mg/kg. An exemplary treatment regime entails
administration twice per week, once per week, once every two weeks,
once every three weeks, once every four weeks, once a month, once
every 3 months or once every three to 6 months. Exemplary dosage
regimens for an anti-hIL20 antibody of the invention include about
1, 3 or 10 mg/kg body weight body weight via intravenous
administration or subcutaneous injection, with the antibody being
given using one of the following dosing schedules: (i) loading
doses every 1-3 weeks for 2-4 dosages, then every two months; (ii)
every four weeks; (iii) every week, or any other optimal dosing. In
some methods, two or more monoclonal antibodies with different
binding specificities are administered simultaneously, in which
case the dosage of each antibody administered falls within the
ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/ml and in some methods about 25-300 .mu.g/ml. The antibody
can alternatively be administered as a sustained release
formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0246] As will be understood by those of ordinary skill in the art,
the appropriate doses of anti-cancer agents will approximate those
already employed in clinical therapies wherein the anti-cancer
agents are administered alone or in combination with other agents.
Variation in dosage will likely occur depending on the condition
being treated. The physician administering treatment will be able
to determine the appropriate dose for the individual subject.
[0247] Articles of Manufacture
[0248] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. For example, the article of
manufacture can comprise a container containing a human or
humanized anti-hIL20 antibody as described herein together with
instructions directing a user to treat a disorder such as an
autoimmune or inflammatory disease or disorder in a human with the
antibody in an effective amount. The article of manufacture
typically comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition that is effective for treating the
condition and may have a sterile access port (for example, the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is the human or humanized
anti-hIL20 antibody herein, or an antigen-binding fragment or
antibody derivative (e.g., an immunoconjugate) comprising such an
antibody. The label or package insert indicates that the
composition is used for treating the condition of choice, such as,
e.g., rheumatoid arthritis, juvenile rheumatold arthritis,
psoriasis, psoriatic arthritis, ankylosing spondylitis, Sjogren's
syndrome, multiple sclerosis, inflammatory bowel disease, systemic
lupus erythematosus, lupus nephritis, or a combination thereof.
[0249] Moreover, the article of manufacture may comprise (a) a
first container with a composition contained therein, wherein the
composition comprises the human or humanized antibody herein, and
(b) a second container with a composition contained therein,
wherein the composition comprises a therapeutic agent other than
the human or humanized antibody. The article of manufacture in this
embodiment of the invention may further comprise a package insert
indicating that the first and second compositions can be used in
combination to treat an autoimmune or inflammatory disease or
disorder. Such therapeutic agents may be any of the adjunct
therapies described in the preceding section. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a pharmaceutically
acceptable buffer, such as bacteriostatic water for injection
(BWFI), phosphate-buffered saline, Ringer's solution and dextrose
solution. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
EXAMPLES
[0250] Further details of the invention are illustrated by the
following non-limiting Examples.
Example 1
Inhibition of IL20-Induced Proliferation by Human Anti-hIL20
Antibodies
[0251] A series of tests were performed to investigate the ability
of human anti-hIL20 monoclonal antibodies to neutralize the effect
of hIL20 induced proliferation of BaF-3 cells transfected with
IL20R1+hIL20R2 (herein "hIL20R").
[0252] Material & Methods
[0253] Media and Buffers.
[0254] Culture medium: Roswell Park Memorial Institute (RPMI 1640)
with Glutamax, 10% heat inactivated foetal bovine serum (FBS), 1%
penicillin/streptomycin (P/S) (BioWhitaker Cat.No. DE17-602E), 1
mg/ml Geneticin (GIBCO Cat.No. 10131-019), 200 .mu.g/ml Zeocin
(Invitrogen 45-0430), 1 ng/ml murine IL-3 (TriChem ApS Cat.No.
213-13), 50 .mu.M 2-Mercapto-ethanol (Gibco Cat.No. 31350-010).
Assay medium: RPMI 1640 with Glutamax, 10% heat inactivated FBS, 1%
P/S (BioWhitaker Cat.No. DE17-602E), 1 mg/ml Geneticin (GIBCO
Cat.No. 10131-019), 200 .mu.g/ml Zeocin (Invitrogen 45-0430), 50
.mu.M 2-Mercapto-ethanol (Gibco Cat.No. 31350-010). AlamarBlue dye:
(BioScource, Dal1100) was used to assess proliferation. The
fluorescence intensity was measured on a spectrofluorometer (bmg
POLARstar+ Galaxy) at excitation 555-12 nm and emission 590 nm.
[0255] Antibodies, Cells and Cytokines.
[0256] Mice were immunized by injecting subcutaneously 20 .mu.g of
human IL-20 in FCA followed by two injections with 20 .mu.g of
hIL20 in FIA. High-responder mice were boosted intravenously with
25 .mu.g of hIL20 and the spleens were harvested after 3 days.
Spleen cells were fused with the myeloma cell line (Kohler, G &
Milstein C. (1976), European J. Immunology, 6:511-19). Supernatants
were screened for human IL-20 antibody production in an indirect
hIL20-specific ELISA, and purified. Rabbit anti-hIL20 polyclonal
antibody (pAb) was produced by immunizing rabbits four times with
50 .mu.g hIL20 at 14 days intervals and five times with 10 .mu.g/ml
hIL20 once monthly. The serum was purified (2313B). BaF-3(hIL20R)
cells were from a BaF-3 cell line transfected with the genes for
hIL20R1 (Zcytor7) and hIL20R2 (Dirs1) and the plasmid KZ134 that
bears luciferase under control of the signal transduction and
transcription protein (STAT)-promotor elements. The BaF-3 cell line
was generated by selection in pyromycin (Zcytor7) and zeozin
(Dirs1) and received from Zymogenetics Institute.
[0257] Stimulation assay.
[0258] An initial stimulation assay was made to assess the level of
hIL20 to be used in the inhibition assay. BaF-3(hIL20R) cells were
washed thoroughly in assay medium to get rid of residual IL-3. The
cells were then seeded into 96-well microtiter plates (flat-well
view plate Packard cat.S00190) at
10.sup.4-5.times.10.sup.4cells/well. Serial dilutions of hIL20
(10.sup.-7M to 10.sup.-13M) were added to the wells and additional
wells with cells but no hIL20 served as negative control. The cells
were cultured for three days in 5% CO.sub.2 at 37.degree. C. For
the last 6 hours of the culture period, 10 .mu.l alamarBlue was
added to each well. The cells were analyzed for fluorescence
intensity on a spectrofluorometer (bmg POLARstar+ Galaxy) at
excitation 555-12 nm and emission 590 nm. For inhibition analysis,
a constant concentration of IL20 was used to stimulate the cells.
This concentration was chosen on basis of approximately 90% of max
stimulation in the proliferation assay which in most of our assays
corresponded to 10.sup.-9M hIL20.
[0259] Inhibition Assay.
[0260] 1.times.10.sup.4-5.times.10.sup.4cells/well of washed
BaF-3(hIL20R) cells were added to microtiter wells in assay medium
and 10.sup.-9M hIL20 (final concentration) was added to each well
except for wells used as negative control containing only cells.
This concentration corresponded to approx. 90% of maximum
stimulation with the hIL20 cytokine. Serial dilutions of antibody
(i.e., 100 .mu.g and 2-fold dilutions) were added to the wells
already containing cells and cytokines (except wells used for
positive controls containing only cells+hIL20). The mixture of
cells, cytokine and antibody were incubated in 100 .mu.l/well for
72 hours in 5% CO.sub.2 at 37.degree. C. The last 6 hours of
incubation included 10 .mu.l/well of alamarBlue. The plates were
analysed for fluorescence intensity on a spectrofluorometer (bmg
POLARstar+ Galaxy) at excitation 555-12 nm and emission 590 nm. The
curves were drawn and the potency (half maximal inhibition
(IC.sub.50)) was calculated using Prism 4 (GraphPad PRISM software
Inc.). Efficacy was calculated as 1-(max inhibition of
antibody/(initial stimulation of cytokine-no stimulation of
cytokine))*100%.
[0261] Results
[0262] An initial stimulation of BaF-3(hIL20R) at 10.sup.-9M hIL20
was chosen from dose response curves. The results from a series of
inhibition assays are shown in Table 1. The potency (IC.sub.50)
varied among the different inhibitory antibodies. 15D2 was one of
the most potent inhibitors, and had the lowest interassay
variation. 15D2 also had a high affinity compared to the polyclonal
rabbit anti-hIL20 antibody preparation.
TABLE-US-00004 TABLE 1 Inhibition of hIL20 induced proliferation
Inhibition of IL20 induced proliferation at 1e-9M hIL20, IC50 (uM)
Anti-IL20 1 2 3 4 5 6 7 2F6 15.4 13.88 1.67 0.36 1.8 0.32 15.38 F56
(LC: F56Type1; 0.04 0.73 HC: F56) F18 (LC: F56Type1; 0.05 HC: F18)
C3 -- -- -- -- 5B7 0.26 4.54 0.76 0.2 15D2 0.18 0.42 0.34 0.12
0.4358 C11 0.26 2.0 0.37 0.1860 F18 (from 0.12 0.09 hybridoma) F56
(from 4.97 hybrodima) 41A6 5.36 3.12 1.32 3.118 41F10 5 4.7 2.55
42A5 0.66 0.21 54F10 2.560 24 4.971
Example 2
Comparative Inhibition of IL20-, IL19- or IL24-Induced
Proliferation
[0263] Human antibody 15D2 was tested for its ability to neutralize
cynomolgus IL20 and mouse IL20. Both mouse and cynomolgus IL20 are
able to induce proliferation in BaF-3(hIL20R) cells. 15D2 was also
tested for inhibition of proliferation induced by hIL19 or hIL24 on
BaF-3(hIL20R). Both hIL24 and hIL19 bind the hIL20R1+hIL20R2
receptor.
[0264] Material & Methods
[0265] Media and Buffers.
[0266] See Example 1.
[0267] Antibodies, Cells and Cytokines.
[0268] See Example 1. Human and cynomolgous IL20 were produced in
E. coli. Mouse IL20 was from BioSource International Inc. Human
IL19 and IL24 were from R&D systems.
[0269] Stimulation Assay.
[0270] An initial stimulation assay was made to asses the initial
concentration of hIL20, cynomolgus IL20 and mouse IL20 to be used
in the inhibition assay, using the same initial stimulation assay
described in Example 1. Initial stimulation with hIL19 and hIL24
was assessed by the same method. It was decided to use three
different initial concentrations of IL19 and hIL24 in the
inhibition assay.
[0271] Inhibition Assay.
[0272] (1) Effect on anti-hIL20 mAb on cynomolgus IL20 and mouse
IL20 induced proliferation:
1.times.10.sup.4-5.times.10.sup.4cells/well of washed BaF-3(hIL20R)
cells were added to microtiter wells in assay medium and,
10.sup.-9M hIL20, cynomolgus IL20 or mouse IL20 (final
concentration) was added to each well except for wells used as
negative controls containing only cells. This concentration
corresponded to approx. 90% of maximum stimulation with the
cytokines. Serial dilutions of antibody 1400-250-15D2 (100 .mu.g
and 2-fold dilutions) were added to the wells already containing
cells and cytokines (except wells used for positive controls
containing only cells+cytokines).
[0273] (2) Effect on anti-hIL20 mAb on hIL19 and hIL24 induced
proliferation:1.times.10.sup.4-5.times.10.sup.4cells/well of washed
BaF-3(hIL20R) cells were added to microtiter wells in assay medium
and three different concentrations of cytokine (10.sup.8M,
10.sup.-9M and 10.sup.-10M final concentration) of either hIL20,
hIL19 or hIL24 was added to each well, except for wells used as
negative controls and containing only cells. This concentration
corresponded to approximately 90% of maximum stimulation with the
cytokines. Serial dilutions of antibody 15D2 (100 .mu.g and 2-fold
dilutions) were added to the wells already containing cells and
cytokines (except wells used for positive controls containing only
cells+cytokines).
[0274] For both assays, the mixture of cells, cytokine and antibody
were incubated in 100 .mu.l/well for 72 hours in 5% CO.sub.2 at
37.degree. C. The last 6 hours of incubation included 10 .mu.l/well
of alamarBlue. The plates were analysed for fluorescence intensity
on a spectrofluorometer (bmg POLARstar+ Galaxy) at excitation
555-12 nm and emission 590 nm. The curves were drawn and the
potency (half maximal inhibition (IC.sub.50)) was calculated using
Prism 4 (GraphPad PRISM software Inc.).
[0275] The efficacy (the percentage inhibition by antibodies) was
calculated as 1-(max inhibition of antibody/(initial stimulation of
cytokine-no stimulation of cytokine))*100%.
[0276] Results
[0277] Effect of anti-hIL20 mAb on cynomolgus IL20 and mouse IL20
induced proliferation.
[0278] When BaF-3(hIL20R) cells were stimulated with human,
cynomolgus or mouse IL20, the cells proliferated and this
proliferation could be inhibited by anti-hIL20 mAb 15D2 in a dose
dependent manner. The efficacy of 15D2 was near 100% at 66 mM IL20
for all three cytokines. The potencies of the antibody could not be
directly compared since the affinity for the hIL20R was different
for the three cytokines.
[0279] Effect of Anti-hIL20 mAb on hIL19 and hIL24 Induced
Proliferation.
[0280] BaF-3(hIL20R) cells were stimulated with three different
concentrations of hIL20, hIL19 or hIL24 (FIG. 5). A dose-dependent
response was detected for inhibition of hIL20 induced proliferation
(FIG. 5A). This experiment served as positive control for the
subsequent hIL19 and hIL24 experiments. When the BaF-3(hIL20R)
cells initially were stimulated with hIL19, 15D2 was not able to
inhibit the proliferation regardless of which of the three initial
concentrations of hIL19 were used (FIG. 5B). The same results were
obtained when the cells initially were stimulated with hIL24 (FIG.
5C).
[0281] Accordingly, 15D2 inhibited cynomolgus IL20-induced as well
as mouse IL20-induced proliferation of the BaF-3(hIL20R) cells,
while hIL19 or hIL24-induced proliferation of BaF-3(hIL20R) was not
inhibited by 15D2.
Example 3
Solubility
[0282] Seven different human IgG4 antibodies against IL20, produced
in HEK293 cells and purified by the same process, were compared for
their ability to reach high concentration in solution, by using
centrifuge filters of the type Amicon Ultra (Millipore Corp, MA)
with a cut-off weight of 50 kD. All samples were centrifuged
according to manufacturer instructions for 1 hour. The initial
concentration of the antibodies ranged from 0.5 to 1.8 mg/ml, and
all antibodies were formulated in 20 mM Na-Phosphate, 150 mM NaCl
at pH 7.4. The VH and VL sequences of the tested antibodies are
shown in FIGS. 4A and 4B, respectively.
[0283] Table 2 shows the concentrations and recoveries obtained
after concentrating human anti-IL20 IgG4 antibodies using Amicon
Ultra 50 kD centrifuge filters. The highest concentration (above
100 mg/ml) was reached for 15D2, followed by 5B7 at above 80 mg/ml,
both of which also had a high recovery. All antibodies retained its
monomeric structure at the higher concentration as seen with
dynamic light scattering analysis, except 2F6, which showed signs
of increased dimerization.
TABLE-US-00005 TABLE 2 Solubility and recovery of human anti-hIL20
antibodies Concentration Anti-IL20 (mg/ml) Recovery (%) 2F6 50 80
F56 (HC: F56; LC: F56type1) 14 48 F18 (HC: F18; LC: F56type1) 31 64
F56/F18 (HC: F56/F18; LC: F56Type1) 24 42 C3 53 80 5B7 84 79 15D2
109 81
[0284] Analysis of the variable region amino acid and/or nucleic
acid sequences of the antibody heavy and light chain variable
regions revealed the germline sequences shown in Table 3. An
additional antibody, C11 (see Example 1) was also sequenced,
revealing a HC having the sequence of SEQ ID NO:25 and the same VL
as F56Type1.
TABLE-US-00006 TABLE 3 VL, VH, and germline sequences of human
anti-IL20 antibodies Germline Light Chain 2F6 (SEQ ID NO: 19)
VKI_L24/JK4 C3 (SEQ ID NO: 21) VKIII_L6/JK2 F56type1, 15D2, and 5B7
(SEQ ID NO: 9) VKI_L18/JK4 F56type2 (SEQ ID NO: 24) VKIII_A27/JK1
Heavy chain 2F6 (SEQ ID NO: 18) VH2_05/D3_10/JH4 C3 (SEQ ID NO: 20)
VH4_39/D_/JH6 F18 (SEQ ID NO: 22) VH1_03/D_/JH4 F56 and F56/F18
(SEQ ID NO: 23) VH1_03/D_/JH4 5B7 (SEQ ID NO: 7) VH1_03/D3_10/JH6
15D2 (SEQ ID NO: 6) VH1_03/D3_10/JH6
Example 4
Binding of Antibodies to hIL20
[0285] A Surface Plasmon Resonance (SPR) experiment was performed
on a BiacoreT100, in order to determine whether individual human
anti-hIL20 antibodies were able to bind simultaneously to
recombinant hIL20. For comparison, three IL20R1/IL20R2-neutralizing
rat anti-hIL20 mAbs, designated 262.4.1.2.2.1, 262.5.1.6.4.4 and
262.7.1.3.2.4 and described in WO2005/052000, were included. An
inability to bind simultaneously indicates common or overlapping
epitopes, though factors such as steric hindrance and
conformational changes may contribute.
[0286] Materials & Methods
[0287] The experiment was performed on a CM5 chip with immobilized
anti-hIL20 antibodies. Each antibody was immobilized on separate
chips to a level of .about.1000 RU by standard amine coupling. All
samples were diluted in running-buffer, HBS-EP pH 7.4 (10 mM HEPES,
150 mM NaCl, 3 mM EDTA and 0.005% Polysorbat P20). Recombinant
hIL20 (10 .mu.g/ml) was injected for 180 s, followed by injection
of 15D2 (10 .mu.g/ml) for 180 s.
[0288] Results
[0289] The human antibodies 15D2 and 5B7 were not able to bind
simultaneously to recombinant hIL20, indicating a common or
overlapping epitope. In contrast, 15D2 was able to bind
simultaneously with each of 262.4.1.2.2.1, 262.5.1.6.4.4 or
262.7.1.3.2.4 mAbs, indicating that the epitope of 15D2 was
different from that of 262.4.1.2.2.1, 262.5.1.6.4.4 and
262.7.1.3.2.4.
Example 5
Binding of Antibodies to Denatured hIL20
[0290] This experiment was conducted to determine whether the human
anti-IL20 antibodies also bound fully denatured antigen. If no
binding activity could be detected, then peptide arrays would not
be appropriate for epitope mapping (see below). In order to test
for binding to the denatured antigen, native and denatured hIL20
preparations were subjected to SDS-PAGE, followed by Western-blot
using 15D2 and 5B7 for detection.
[0291] Materials & Methods
[0292] Recombinant hIL20 was denatured by boiling for 10 min. in
sample buffer (NuPage, Invitrogen) containing 50 mM DTT. The
samples were run in SDS-PAGE (20 ul/well including sample-buffer)
and blotted to a nitrocellulose membrane. The membrane was
incubated with the primary mAb (10 .mu.g/ml in blotting buffer
(Novex, Invitrogen)), followed by incubation with HRP-conjugated
Rabbit anti-human IgG polyclonal Ab (DAKO). Bands were visualized
using TMB-substrate (Kem-En-Tech).
[0293] Results
[0294] Both 15D2 and 5B7 recognized the native- and denatured form
of the antigen, indicating that the epitope is continuous
(linear).
Example 6
Surface Plasmon Resonance Analysis of Peptide Binding
[0295] An SPR experiment was performed in order to determine
whether the anti-hIL20 antibodies 15D2, 262.4.1.2.2.1,
262.5.1.6.4.4 or 262.7.1.3.2.4 bound to the region corresponding to
residues 42-102 of the unprocessed precursor of hIL20 (SEQ ID
NO:2), corresponding to residues 18-78 of mature hIL20 (SEQ ID
NO:1). The antibodies were shown to recognize both the native and
denatured form of hIL20 (for 15D2, see above).
[0296] Materials & Methods
[0297] Five 20mer peptides with a frame shift of 10 residues were
synthesized:
TABLE-US-00007 (residues 18-37 of SEQ ID NO: 1) 1
IRNGFSEIRGSVQAKDGNID (residues 28-47 of SEQ ID NO: 1) 2)
SVQAKDGNIDIRILRRTESL (residues 38-57 of SEQ ID NO. 1) 3)
IRILRRTESLQDTKPANRSS (residues 48-67 of SEQ ID NO. 1) 4)
QDTKPANRSSLLRHLLRLYL (residues 58-78 of SEQ ID NO: 1) 5)
LLRHLLRLYLDRVFKNYQTPD
[0298] The peptides, containing N-terminal biotin, were immobilized
(500 RU) in individual flow cells on streptavidin coated (SA)
chips. The individual antibodies (5 .mu.g/ml) were injected across
all flow cells for 120 s, followed by a 180 s dissociation phase.
Experiments were performed on Biacore3000 and BiacoreT100
instruments. The results were evaluated using Scrubber2 software
(BioLogic Software Pty Ltd).
[0299] Results
[0300] No 15D2, 262.5.1.6.4.4 or 262.7.1.3.2.4 binding to the
peptides was detected. The rat anti-hIL20 mAb 262.4.1.2.2.1
demonstrated binding to peptide 4 and 5, indicating binding to
their shared sequence LLRHLLRLY. All mAbs demonstrated binding to
immobilized intact biotinylated IL20.
Example 7
Primary Peptide Array ("Peptide Walk")
[0301] A peptide array consisting of 18mer hIL20 peptides with a
frame shift of 4 residues was made and screened against
fluorescence-labelled anti-hIL20 antibodies 15D2 or 5B7.
[0302] Materials and Methods
[0303] Synthesis of Epitope Arrays.
[0304] The epitope mapping arrays were synthesized on cellulose
sheets (Aims-Scientific, Germany) on an array synthesizer (Multipep
Spot, Intavis, Germany) essentially using the protocols provided
from the manufacturer. Fmoc-amino acids were purchased from
Novabiochem (Germany) and dissolved in N-methylpyrrolidinone (NMP)
containing 0.3 M Hydrobenzotriazole (HOBt) to a final concentration
of 0.3M. Coupling was done by activating with
diisopropylcarbodiimide (DIC), and deprotection of the Fmoc group
was done by 20% piperidine in NMP. The individual sequences were
designed by the software accompanying the array synthesiser. After
synthesis the protecting groups were removed by treating the sheets
with trifluoroacetic acid (TFA) 95% containing triisopropylsilane
(TIPS) for 60 min. Then washed with dichloromethane (DCM) and
N-methylpyrrolidinone (NMP) and finally with water.
[0305] Labelling of Antibodies.
[0306] The screenings were done using fluorescence labelled
antibodies. The labelling was done by gel-filtering the antibody
stock against 1% NaHCO3, using NAP5 column (GE Healthcare according
the manual from manufacturer. This was followed by adding 25 mole
equivalents of 5(6)-carboxyfluorescein N-hydroxysuccinimide ester
(Sigma, C1609) dissolved in DMSO. The coupling was allowed to
continue for 2 hours followed by a gelfiltration against TRIS
washing buffer (50 mM TRIS, pH=7.4, 0.15M NaCl, 0.1 M ArgHCl, 0.05%
Tween 20) in order to removed uncoupled fluorescein.
[0307] Screening and Analyzing Arrays.
[0308] Screening of arrays were done by adding 10 .mu.l of antibody
to 30 ml incubation buffer (0.5% BSA, 50 mM TRIS, pH=7.4, 0.15M
NaCl, 0.1 M ArgHCl, 0.05% Tween 20). The sheets were incubated for
1-2 hours, followed by washing five times with washing buffer. Then
the sheets were scanned using a laser scanner (Typhoon 9410, GE
Healthcare) and the image file (.gel format) was analysed using
dedicated array software ArrayPro Analyzer (Media Cypernetics,
USA). The fluorescence intensity were measured and transformed into
digits that were exported to Prism 5 (Graph Pad Software, USA) for
further analysis.
[0309] Results
[0310] The results from the primary peptide array analysis, shown
in FIG. 6, clearly identified that 15D2 and 5B7 both bind a linear
epitope located in the region corresponding to residues 73-96 in
mature hIL20 (SEQ ID NO:1), corresponding to residues 97-120 of the
precursor (SEQ ID NO:2).
Example 8
Secondary Peptide Array Analysis--Terminal Deletions
[0311] In order to narrow down the length of the epitope and to
evaluate which residues were important for the binding of anti-IL20
antibodies 15D2 and 5B7, an array of various truncations was made.
An ala-scan was also included (section 5).
[0312] For Materials and Methods, see Example 7.
[0313] Results
[0314] The truncations from the hIL20 C-terminal revealed a gradual
reduction in 15D2 binding from peptides 4 to 7 (FIG. 7A). A more
sudden decline in binding activity was seen for the N-terminal
truncations, where removal of D (Asp) and H (His) dramatically
reduced the affinity. Overall, the results showed that the minimum
epitope had the sequence DHYTLRKISSLANSFL, corresponding to
residues 78 to 93 of SEQ ID NO:1.
[0315] For 5B7, the truncations revealed a sudden reduction in
binding when deleting from the N-terminal (FIG. 7B). A decline of
about 50% in binding activity was seen for the N-terminal
truncations when removing D (Asp), and removing H (His)
dramatically reduced the affinity to no detectable binding.
Removing from the C-terminal resulted in a less sudden decline in
affinity until removing N (Asn). Overall, the results indicate that
the minimum 5B7 epitope had the sequence DHYTLRKISSLAN (residues
78-90 of SEQ ID NO:1), although a longer epitope,
DHYTLRKISSLANSFLTIK (residues 78-96 of SEQ ID NO:1), did present a
higher affinity. This indicated the presence of some structural
requirements, e.g., .alpha.-helix dependence.
[0316] The result of the Ala scan clearly indicated that, for 15D2,
three residues, H79 (His-79), R83 (Arg-83) and N90 (Asn-90) of SEQ
ID NO:1 were most critical for binding (FIG. 8A). Also sensitive,
but to a lesser extent, were residues D78 (Asp-78), S86 (Ser-86),
F92 (Phe-92) and L93 (Leu-93). The residues H79 and N90 were most
critical also for 5B7 binding, while R83 (Arg) appeared fairly
critical (FIG. 8B).
[0317] In conclusion, the human anti-hIL20 antibodies 15D2 and 5B7
both bind a linear epitope (functional) of similar length and
specificity, shown in FIG. 1. Residues H79, R83 and N90 (bold and
single-underlined) of mature hIL20 (SEQ ID NO:1); corresponding to
H103, R107, and N114 in hIL20 precursor (SEQ ID NO:2), were found
the most critical, with residues D78, S86, F92 and L93 (bold and
double-underlined) being moderately critical. The main difference
between the two antibodies was that R83 was slightly less critical
for 5B7 binding as compared to 15D2 binding.
[0318] The position of the 15D2/5B7 epitope and the location of the
most critical residues were revealed using the crystal structure of
the homologous protein IL19 (Chang et al. J Biol Chem 2003; 278:
3308). It was found that the location of the epitope corresponded
to helix E in IL19, and that the most critical residues in hIL20;
H79, R83, and N90 of SEQ ID NO:1, are exposed to the solvent. Note
that the hIL20 residue H79 is a proline in hIL19.
Example 9
Neutralization of IL20 Activation of IL20R1/IL20R2 and
IL22R1/IL20R2 Receptor Complexes
[0319] This Example shows that human antibody 15D2 is capable of
neutralizing murine, cynomolgous, and hIL20 activation of
recombinantly expressed IL20R1/IL20R2 and IL22R1/IL20R2 receptor
complexes.
[0320] Materials and Methods
[0321] Cloning of IL20 receptors.
[0322] Human IL20 receptors, IL22R1 (EMBL BCO29273), IL20R1 (EMBL
AF184971) and IL20R2 (EMBL AY358305) were PCR-amplified from NHEK
(Normal Human Epidermal Keratinocyte) cDNA and cloned into
pcDNA3,1+(zeocin) (IL22R1 and IL20R1) and pcDNA3,1+(hygro)(IL20R2).
Mouse IL20 receptors, mIL22R1 (EMBL AY103454), mIL20R1 (EMBL
AK054215) and mIL20R2 (EMBL BC107264) were cloned by PCR using
mouse liver, testis and skin cDNA, respectively, as templates. Each
of these IL20R sequences is incorporated by reference, in its
entirety. The mIL22R1 and mIL20R1
[0323] PCR-product were cloned into pcDNA3,1+(zeo) and mIL20R2 into
pcDNA3,1+(hygro). Cynomolgus IL20 receptors, cynolL22R1 (SEQ ID
NO:28), cynolL20R1 (SEQ ID NO:26) and cynolL20R2 (SEQ ID NO:27)
were cloned by PCR using cynomolgus skin cDNA. The cynolL20R1 and
cynolL22R1 receptors were inserted into pcDNA3,1+(zeocin) and
cynolL20R2 was inserted into pcDNA3,1+(neomycin).
[0324] The following 5' and 3' primers were used for
PCR-amplification of coding cDNA.
TABLE-US-00008 Human IL22R1: (SEQ ID NOS: 29 and 30)
agaattccaccatgaggacgctgctgacca and gctcgagacagggaggaagcac-caag
Human IL20R1: (SEQ ID NOS: 31 and 32) cgaattcccttggtttctggggaag and
gctcgagcacaggaaacaaaaggcaaa Human IL20R2: (SEQ ID NOS: 33 and 34)
agaattctggaaagaaacaatgttctaggtcaa and gctcgagcttcacctgggcccttcc
Murine IL22R1: (SEQ ID NOS: 35 and 36)
ccgaattcgccaccatgaagacactactgaccatc and
cttgcggccgctcag-gattcccactgcacagtc Murine IL20R1: (SEQ ID NOS: 37
and 38) ttgaattcgccaccatgcacactcccggga and
ttgcggccgcctagctttccatttgta-catgtaacc Murine IL20R2: (SEQ ID NO: 39
and 30) ttggatccgccaccatgatttcccagggagtctg and
ttgcggccgctcaagtctgtga-gatccagac Cynomolgus IL22R1: (SEQ ID NO: 41
and 42) agaattccaccatgaggacgctgctgacca and
gctcgagacagggaggaag-caccaag Cynomolgus IL20R1: (SEQ ID NOS: 43 and
44) gtgggactgagcagtctgctg and aggcaaaaggaagtgttggca Cynomolgus
IL20R2: (SEQ ID NOS: 45 and 46) agaattctggaaagaaacaatgttctaggtcaa
and gctcgagcttcac-ctgggcccttcc
[0325] STAT-reporter KZ136 is a luciferase reporter containing
STAT-elements and a Serum Response Element (SRE) (Poulsen-L K et
al. J Biol Chem 1998; 273:6228-6232).
[0326] Luciferase Assay in Transient Transfection.
[0327] Day 0: BHK cells were seeded in T80 flask to a confluency of
40%. Day 1: 7.5 micrograms DNA was transfected using 36 microliters
FuGene (Roche Applied Science) according to manufacturers manual,
2.5 micrograms of receptor chain 1 (IL20R1 or IL22R1) and 2.5
micrograms of chain 2 (IL20R2) and 2.5 micrograms of luciferase
reporter (KZ136). Day 2: The cells were detached using Versene and
20,000 cells per well were seeded in a black view plate. After the
cells have reattached the well surface the media was exchanged with
serum-free media. Day 3: either 20 microliter of 10 mM IL20 or 20
microliter of serum-free medium was added to the wells. Four hours
later the luciferase activity was determined; media was removed and
100 microliter 1.times.PBS was added to each well followed by the
addition of 100 microliter Luclite substrate (PerkinElmer). The
plate was incubated for 30 minutes. The luminescence was detected
by a Topcount NXT (Perkin Elmer). IL20 used here are either
recombinantly produced hIL20 produced in E. coli, murine IL20
purchased from Biosource, #PMC0201, or cynomolgus IL20 produced by
transient expression in HEK293 6E cells and purified.
[0328] Results
[0329] Neutralization of Murine IL20.
[0330] The murine IL20 receptors were cloned and a transient
luciferase reporter assay was set up in BHK cells. Both the two
human receptor complexes and the two murine receptor complexes were
stimulated with 1 nM murine IL20. Murine IL20 could activate both
human and murine IL20R1/IL20R2 and IL22R1/IL20R2 receptor
complexes. Neutralization by 15D2 was tested on one of the receptor
complexes. The murine IL20R2/IL22R1 complex was transfected into
BHK cells together with the STAT3 reporter construct. The receptor
complex was stimulated with 1 nM murine IL20 and exposed to 15D2
antibody in 1, 10 and 50 microgram/ml doses (FIG. 9). The lowest
dose, 1 microgram/ml, neutralized the effect almost totally.
[0331] Neutralization of Cynomolgus IL20.
[0332] Cynomolgus IL20 receptor sequences IL20R1, IL20R2 and IL22R1
were cloned from cynomolgus skin tissue cDNA, using oligonucleotide
primers based on human sequence. The respective sequence identities
between cynomolgus and human receptor sequences were 96.8% for
IL20R1, 98.9% for IL20R2, and 95.5% for IL22R1. BHK cells were
transfected with the two receptor complexes, IL20R1/IL20R2 or
IL22R1/IL20R2 together with the KZ136 (the STAT3 luciferase
reporter) plasmid. The cynomolgus IL20R2/IL22R1 complex was induced
3-4 fold using 1 nM cynolL20. The IL20R1/IL20R2 complex from
cynomolgus was stimulated about 2-fold. Increasing the amount of
15D2 decreased the IL20 activity, showing that 15D2 could
neutralize cynomolgous IL20 (FIG. 10).
[0333] Neutralization of hIL20.
[0334] Human IL20 receptors IL20R1, IL20R2 and IL22R1, were cloned
from NHEK (Normal Human Epidermal Keratinocyte) cDNA. Expression
plasmids encoding the IL20 receptors were transiently transfected
into BHK cells together with the luciferase reporter vector, KZ136.
The stimulation was done using 1 nM hIL20 and an induction of about
3 fold was seen. Increasing the amount of 15D2 antibody decreased
the IL20 activity (FIG. 11).
Example 10
Inhibition of hIL20-Induced Proliferation
[0335] This Example evaluates the inhibitory effects of 15D2 and
rat anti-hIL20 mAbs on IL20-induced proliferation of BaF-3 cells
expressing IL20R1 and IL20R2 (herein "hIL20R"), and the form of
IL20 bound by 15D2.
[0336] Materials & Methods
[0337] Three independent experiments ("148", "149" and "150")
compared EC50 values of the four tested abs. First, antibodies were
added in a 3-fold serial dilution with 50 ul/well in media. At the
same time 10 .mu.l hIL20 was added to every well and at last 40
.mu.l of cell-suspension. Antibodies were diluted to a 3-fold
serial dilution (100+200), with the first dilution in the assay at
20 .mu.g/ml. hIL20 preparation was diluted to 10.sup.-7M, with
dilution in assay to 10.sup.-8M. As a control, the following
stimulation curve was made: hIL20 was diluted to 10.sup.-6M and
from this a 10-fold dilution row. First dilution in the assay was
10-7M.
[0338] BaF-3 cells recombinantly expressing human IL20R1/IL20R2
were centrifuged, resuspended in media without IL3, and counted.
They were then washed twice in media without IL3, and added into
96-wells flat-bottomed view plates at a concentration of
10.sup.4c/well and 40 .mu.l. Dilution of cells: 10.sup.4c/40 .mu.l,
2.5.times.10.sup.6c/10 ml (enough for 2 plates). Incubation of
plates for 3 days in a CO.sub.2 incubator (5% CO.sub.2, 37.degree.
C.). AlamarBlue 10 .mu.l/w was added, and after 6 hours incubation
plates were measured on a Polarstar fluorometer, exitation 550-12
nm and emission 590 nm.
[0339] In order to investigate whether 15D2 bound the soluble form
of IL20 or the receptor-bound form of IL20, an experimental setup
was designed where 15D2 was added in a 3-fold serial dilution. At
the same time, 10 .mu.l hIL20 was added to the wells and
subsequently 40 .mu.l of BaF3(hIL20R=hIL20R1 and hIL20R2)
cell-suspension. Finally, hIL19 was added simultaneously (t=0) or
after 1 minute (t=1).
[0340] Results
[0341] From the initial experiment testing proliferation as a
function of concentration of hIL20 shown, it was chosen to measure
the EC50 values of antibodies at 10.sup.-8 M hIL20.
[0342] Three independent experiments tested the inhibitory effects
of antibodies on IL20 induced proliferation. EC50 values were
calculated both with and without defined top and bottom. The latter
gave the best correlation between the three independent experiments
and was used for comparison of inhibitory effects (curves were
fitted by "sigmoidal dose-response with variable slope" in Prism).
Table 4 below shows the EC50 values of the three experiments,
comparing EC50 values for inhibition of IL20-induced proliferation
of BaF-3 cells expressing IL20R1 and IL20R2.
TABLE-US-00009 TABLE 4 Inhibition of hIL20-induced proliferation of
BaF-3 (hIL20R1/hIL20R2) cells Anti-IL20 ab EC50 (nM) 15D2 6.5, 6.7,
6.8* 6.6 13.07 262.4.1.2.2.1 3.4 4.0 2.77 262.5.1.6.4.4 17.0 25.6
14.53 262.7.1.3.2.4 4.3 4.2 4.16 *Included on three plates
[0343] The EC50 values were analyzed by one-way ANOVA with Tukey
post test. All of 15D2, 262.4.1.2.2.1 and 262.7.1.3.2.4 inhibited
IL20 induced proliferation in IL20R transfected BaF-3 cells
significantly better than 262.5.1.6.4.4. While the average EC50
values for 262.4.1.2.2.1 and 262.7.1.3.2.4 were lower than those of
15D2, the differences were not statistically significant
(P<0.05). With respect to the assay investigating whether 15D2
bound the soluble or receptor-bound form of IL20, the results
showed that addition of IL19 reverted the 15D2 blocking of
IL20-induced proliferation (FIG. 12). This meant that 15D2
prevented binding of IL20 to the hIL20R, but that 15D2 did not
prevent binding of IL19 to the receptor. Thus, 15D2 bound the
soluble form of IL20 and not the receptor-bound form, which would
otherwise block access of IL19 to the receptor.
Example 11
Neutralization of IL20 Activation of IL22R1/IL20R2
[0344] This example shows the inhibitory effect of human antibody
15D2 and rat anti-hIL20 mAbs on IL20-induced signalling via human
IL22R1/IL20R2 receptor complex in a Luciferase reporter assay.
[0345] Materials and Methods
[0346] Generation a Human IL20 Reporter Cell Line.
[0347] The human IL20R2 in pcDNA3,1+(hygro) plasmid, IL22R1 in
pcDNA3,1+(zeocin)plasmid and the STAT3 reporter plasmid KZ136
(neomycin) were transfected into BHK cells and selected by 200
ug/ml hygromycin, 400 ug/ml zeocin and 600 ug/ml geneticin. Clones
were picked and the most IL20 responsive clone was selected, "BHK
1-B4". The BHK 1-B4 cell line responds approximately 10-fold in
luciferase read-out to 10 nM IL20 compared to basal level (no
stimulation).
[0348] Luciferase Assay in Stable BHK 1-B4 Cells.
[0349] The BHK 1-B4 were seeded in 96 well plates, 20,000
cells/well. Sixteen hours after seeding the cells were stimulated
with 10 nM IL20 or mixtures with 10 nM IL20 and antibodies or plain
media. The cells were stimulated for 4 hours, media was removed,
100 ul PBS (including Ca++ and Mg++) and 100 ul luciferase
substrate (Steady-GLO) was added to the wells. The plate was
incubated for 30 minutes. The luminescence was detected by a
Topcount NXT (PerkinElmer).
[0350] Results
[0351] Neutralization of hIL20 by Anti-IL20 Antibodies.
[0352] The stable cell line BHK 1-B4 was generated by stably
transfection of plasmids expressing IL20R2 and IL22R and the STAT3
Luciferase reporter plasmid, KZ136. Antibodies were mixed with IL20
prior to addition to the cells. The IL20 concentration was kept at
10 nM whereas the antibodies were added in range from 133 nM to
0.19 nM in 3-fold dilutions. Dose-response curves were obtained for
the four antibodies, and EC50 values were calculated based on the
curves fitted by "sigmoidal dose-response with variable slope" in
GraphPad Prism (Table 4). 15D2 neutralized signalling via
hIL22R1/hIL20R2 as efficiently as 262.4.1.2.2.1 and better than
262.5.1.6.4.4 and 262.7.1.3.2.4.
TABLE-US-00010 TABLE 5 Neutralization of IL20 activation of
IL22R1/IL20R2 receptor complex Anti-IL20 antibody EC50 (nM) 15D2
(NN) 4.98 262.4.1.2.2.1 (ZGI) 4.92 262.5.1.6.4.4 (ZGI) 18.33
262.7.1.3.2.4 (ZGI) 11.17
Example 12
Determination of Kinetic Parameters
[0353] Protein interactions can be monitored in real time using
surface plasmon resonance (SPR) analyses. This Example describes
SRP analysis on Biacore 3000 and Biacore T100 instruments, in order
to characterize hybidoma-produced and/or recombinantly expressed
human anti-IL20 antibodies 15D2 and 5B7 with respect to affinity
towards recombinant hIL20.
[0354] Affinity studies were performed using a direct binding
procedure, with the monoclonal antibody covalently coupled via free
amine groups to the carboxymethylated dextrane membrane (CM5) on
the sensor chip surface. Recombinant hIL20 was injected in various
concentrations, followed by a dissociation period with constant
buffer flow over the sensor chip surface. Using this experimental
design, the binding of hIL20 to the immobilized monoclonal antibody
could be regarded as a 1:1 binding, with one hIL20 molecule binding
to one antibody binding site. The kinetic parameters for the
interaction could be calculated using a 1:1 interaction Langmuir
fitting model.
[0355] Materials & Methods
[0356] 15D2 wild-type (wt) expressed by hybridoma cells, 15D2-wt
expressed in HEK293 cells, 15D2 with an S241P mutation expressed in
HEK293 cells, 15D2-S241P expressed in CHO cells, and 5B7-wt
expressed by hybridoma cells, were analyzed. The purified
monoclonal antibodies were immobilized in individual flow cells on
a CM5 type sensor chip. Immobilizations were performed using a
standard amine coupling procedure, aiming for an immobilization
level of 1000 Resonance Units (RU). The antibodies were diluted to
5 .mu.g/ml in 10 mM NaAc pH 5.0. HPS-EP pH 7.4 (10 mM HEPES, 150 mM
NaCl, 3 mM EDTA and 0.005% Polysorbate P20) was used as running
buffer, and diluent for the recombinant hIL20. Recombinant purified
hIL20 was diluted to 100, 50, 25, 12.5, 6.25 and 3.125 nM.
Association (injection) was 3 min., followed by a 30 min.
dissociation (wash) period. Flow rate was 30 .mu.l/min. Experiments
were performed at 25.degree. C. Regeneration of the surface
following each cycle, was accomplished by injection of 30 sec.
pulse of 10 mM Glycin-HCl pH 1.8, at a 30 ul/min flow rate.
Detection in all flow cells simultaneously. Flow cell No. 1
contained no immobilized antibody, and was used for subtraction of
background and bulk. The kinetic parameters were calculated by
local fitting of the data for a given antibody-antigen combination
using a 1:1 Langmuir binding model. Data was inspected for
mass-transport limitations prior to calculation of the kinetic
parameters. Experiments were performed on Biacore 3000 and T100
instruments. Data was evaluated using Biaeval 4.1 and Biacore T100
evaluation software.
[0357] Results.
[0358] The calculated affinities for the binding of recombinant
hIL20 to the individual antibodies are listed in Table 6 below,
showing rate constants and affinities of the individual anti-IL20
monoclonal antibodies. The affinities are listed in molar units
(M), the on-rates in (1/Ms) and the off-rates in (1/s). The rate
constants are listed in brackets below the affinity, as
(on-rate/off-rate).
[0359] The affinity determination, valid for the buffer used and
with the recombinant form of the antigen, demonstrated KD values of
both 15D2-wt HEK293 and 15D2-S241P CHO in the lower pM range.
Furthermore, the affinities of the hybridoma expressed antibodies
demonstrated affinities of about 0.5 nM KD of the 15D2-wt and
5B7-wt.
TABLE-US-00011 TABLE 6 kinetic parameters for 15D2 and 5B7
interactions with recombinant hIL20 KD (M) KD (M) KD (M) Antibody
Hybridoma HEK293 CHO 15D2-wt 5.5E-10 3.2E-11 -- (1.9E+05/ (1.9E+06/
1.1E-04) 6.3E-05) 15D2-S241P -- 3.6E-11 3.1E-11 (1.7E+06/ (2.3E+06/
6.8E-05) 7.1E-05) 5B7 7.5E-10 -- -- (1.4E+05/ 1.0E-04)
Example 13
15D2 Binding Interface on IL20
[0360] This Example identifies the 15D2 binding interface on hIL20
using HX-MS technology. Unless otherwise indicated, the numbering
of hIL20 amino acid residues in this Example refers to SEQ ID NO:1
with an N-terminal Met (M) residue (i.e., residue Y67 in this
Example corresponds to residue Y66 in SEQ ID NO:1).
[0361] HXMS provides the possibility for mimicking in vivo
conditions (see, e.g., Wales and Engen, Mass Spectrom. Rev. 25, 158
(2006), and Coales et al, Rapid Commun. Mass Spectrom. 23, 639
(2009)). The HX-MS technology used here provided information on
which surface exposed amide hydrogens in IL20 became shielded from
exchange with solvent upon 15D2 antibody binding, thereby
facilitating a mapping of the binding interface. Furthermore, the
methodology can also reveal more indirect structural effects in
IL20. Here observed as a slight stabilization of the structure upon
15D2 binding as seen in some regions.
[0362] Amide hydrogen/deuterium exchange (HX) was initiated by a
23-fold dilution of IL20 in the presence or absence of 15D2 (Fab
fragment) into the corresponding deuterated buffer (i.e. 25 mM MES,
80 mM NaCl, 96% D.sub.2O, pH 6.4 (uncorrected value)).
Non-deuterated controls were prepared by dilution into an identical
protiated buffer. All HX reactions were carried out at 20.degree.
C. and contained 4 .mu.M IL20 in the absence or presence of 5 .mu.M
15D2. Preliminary data had demonstrated full saturation of IL20
binding at these protein concentrations.
[0363] At appropriate time intervals, aliquots of the HX reaction
were quenched by an equal volume of ice-cold quenching buffer
(1.35M Tris(2-carboxyethyl)phosphine hydrochloride, adjusted to pH
2.5 using NaOH) resulting in a final pH of 2.6 (uncorrected value).
Quenched samples were immediately injected onto a cooled ultra high
pressure liquid chromatography (UPLC)-mass spectrometry system
(described in detail below) for pepsin digestion, rapid desalting
and mass analysis.
[0364] All sample preparation, handling and injections were
performed by a HD-x PAL auto-sampler (LEAP Technologies Inc.). The
protein and quench solutions were held at 2.degree. C. and
deuterated buffer and labelling reactions were held at 20.degree.
C. A cooling box, temperature controlled at 1.5.degree. C.,
contained the injection and switching valves, tubing, plumbing and
columns. Pepsin column (Applied Biosystems), VanGuard C18 trapping
column (Waters) and Acquity HPLC BEH C18 1.7 um, 2.1.times.100 mm
analytical column (Waters Inc) were used. The LC flow was delivered
from an Acquity HPLC pump using 0.1% Formic acid in H2O and 0.1%
Formic acid in acetonitrile. A Q-ToF premier was used for mass
analysis (Waters Inc).
[0365] Peptic peptides were identified in separate experiments
using standard MS/MS methods. Average masses of peptide isotopic
envelopes were determined from lockmass-corrected centroided data
(processed using MassLynx software, Waters Inc.) using the software
HXExpress (Weis et al., J. Am. Soc. Mass Spectrom. 17, 1700
(2006)).
[0366] The HX time-course of 22 peptides, covering 93% of the
primary sequence of IL20, were monitored in the presence and
absence 15D2 (FIG. 13). The IL20 exchange pattern observed could be
divided into two different groups. One group of peptides displayed
an exchange pattern that was largely unaffected by the binding of
15D2. For example, Peptide 127-145 represented a region of IL20
that was unaffected by 15D2 binding. Some, however, showed a slight
decrease in exchange at 30 sec due to slight stabilization of the
protein structure upon 15D2 binding. For example, Peptides 17-38,
60-66 and 146-153 represented regions of IL20 that were outside the
binding epitope but might show a slight structural stabilization
upon 15D2 binding. In contrast, another group of peptides IL20 show
strong protection, here more than 3 deuterons, from exchange upon
15D2 binding. Peptides 67-83, 67-93, 69-89, 69-93 and 84-93
represented peptides that were part of the binding epitope of 15D2
Thus, the region displaying protection upon 15D2 binding
encompassed peptides from residues 67-93. For example at 30 sec
exchange with D.sub.2O, approximately 10 amides were protected from
exchange in the region 69-93 upon 15D2 binding. The specific
peptides and number of deuterons shielded from exchange upon 15D2
binding are depicted in FIG. 14 where the information gained after
30 sec exchange in D.sub.2O was sub-localized to few residues.
[0367] The 15D2 binding interface could thus be localized to
residues 71-93, containing the sequence VFKNYQTPDHYTLRKISSLANSF and
corresponding to residues 70-92 of SEQ ID NO:1. The region
containing residues 71-83, however, was protected to a lesser
extent from deuterium exchange upon 15D2 binding. This indicates
that the overall 15D2 binding in this region was less tight and,
most likely, that only a fraction of these residues were involved
in 15D2 binding. The residues 84-85 showed complete protection from
exchange upon 15D2 binding and the region 86-93 was also highly
affected by 15D2 binding, together corresponding to residues 83-92
of SEQ ID NO:1.
[0368] All references, including publications, patent applications
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference was individually and
specifically indicated to be incorporated by reference and was set
forth in its entirety herein.
[0369] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way,
[0370] Any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0371] The terms "a" and "an" and "the" and similar referents as
used in the context of describing the invention are to be construed
to cover both the singular and the plural, unless otherwise
indicated herein or clearly contradicted by context.
[0372] Recitation of ranges of values herein are merely intended to
serve as a shorthand method of referring individually to each
separate value falling within the range, unless otherwise indicated
herein, and each separate value is incorporated into the
specification as if it were individually recited herein. Unless
otherwise stated, all exact values provided herein are
representative of corresponding approximate values (e.g., all exact
exemplary values provided with respect to a particular factor or
measurement can be considered to also provide a corresponding
approximate measurement, modified by "about," where
appropriate).
[0373] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context.
[0374] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise indicated. No language in
the specification should be construed as indicating any element is
essential to the practice of the invention unless as much is
explicitly stated.
[0375] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability and/or enforceability of such patent
documents,
[0376] The description herein of any aspect or embodiment of the
invention using terms such as "comprising", "having", "including"
or "containing" with reference to an element or elements is
intended to provide support for a similar aspect or embodiment of
the invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements,
unless otherwise stated or clearly contradicted by context (e.g., a
composition described herein as comprising a particular element
should be understood as also describing a composition consisting of
that element, unless otherwise stated or clearly contradicted by
context).
[0377] This invention includes all modifications and equivalents of
the subject matter recited in the aspects or claims presented
herein to the maximum extent permitted by applicable law.
EXEMPLARY EMBODIMENTS
[0378] The following are exemplary and non-limiting embodiments of
the invention. [0379] 1. An isolated anti-human IL20 antibody or an
antigen-binding fragment thereof, which reduces IL20 mediated
activation of IL20R1/IL20R2 and IL22R1/IL20R2 receptor complexes.
[0380] 2. The antibody or antigen-binding fragment of embodiment 1,
which reduces human IL20 mediated activation of human IL20R1/IL20R2
and IL22R1/IL20R2 receptor complexes. [0381] 3. The antibody or
antigen-binding fragment of any of the preceding embodiments, which
reduces cynomolgus IL20 mediated activation of cynomolgus
IL20R1/IL20R2 and IL22R1/IL20R2 receptor complexes. [0382] 4. The
antibody or antigen-binding fragment of any of the preceding
embodiments, which reduces murine IL20 mediated activation of
murine IL20R1/IL20R2 and IL22R1/IL20R2 receptor complexes. [0383]
5. The antibody or antigen-binding fragment of any of the preceding
embodiments, which reduces the binding of IL20 to the IL20R1/IL20R2
and/or IL22R1/IL20R2 receptor complexes. [0384] 6. The antibody or
antigen-binding fragment of any of the preceding embodiments, which
reduces the binding of IL20 to IL20R2. [0385] 7. The antibody or
antigen-binding fragment of any of embodiments 1-4, which does not
reduce binding of IL19 or IL24 to IL20R1/IL20R2 or IL22R1/IL20R2
receptor complexes. [0386] 8. The antibody or antigen-binding
fragment of any of the preceding embodiments, which binds to an
epitope comprising at least one residue selected from D78-H103 of
mature human IL20 (SEQ ID NO:1), optionally excluding D78. [0387]
9. The antibody or antigen-binding fragment of embodiment 8,
wherein the epitope comprises at least one residue selected from
D78-K96. [0388] 10. The antibody or antigen-binding fragment of
embodiment 9, wherein the epitope comprises at least one residue
selected from D78-L93 or R83-F92. [0389] 11. The antibody or
antigen-binding fragment of embodiment 10, wherein the epitope
comprises at least one residue selected from H79-N90. [0390] 12.
The antibody or antigen-binding fragment of embodiment 8, wherein
the epitope comprises at least 3 residues selected from D78-H103.
[0391] 13. The antibody or antigen-binding fragment of embodiment
12, wherein the epitope comprises at least 3 residues selected from
D78-K96. [0392] 14. The antibody or antigen-binding fragment of
embodiment 13, wherein the epitope comprises at least 3 residues
selected from D78-L93 or R83-F92. [0393] 15. The antibody or
antigen-binding fragment of embodiment 14, wherein the epitope
comprises at least 3 residues selected from H79-N90. [0394] 16. The
antibody or antigen-binding fragment of embodiment 8, wherein the
epitope comprises at least 5 residues selected from D78-H103.
[0395] 17. The antibody or antigen-binding fragment of embodiment
16, wherein the epitope comprises at least 5 residues selected from
D78-K96 or R83-F92. [0396] 18. The antibody or antigen-binding
fragment of embodiment 17, wherein the epitope comprises at least 5
residues selected from D78-L93. [0397] 19. The antibody or
antigen-binding fragment of embodiment 18, wherein the epitope
comprises at least 5 residues selected from H79-N90. [0398] 20. The
antibody or antigen-binding fragment of embodiment 8, wherein the
epitope is in the segment corresponding to residues D78-H103.
[0399] 21. The antibody or antigen-binding fragment of embodiment
20, wherein the epitope is in the segment corresponding to residues
D78-K96. [0400] 22. The antibody or antigen-binding fragment of
embodiment 21, wherein the epitope is in the segment corresponding
to residues D78-L93. [0401] 23. The antibody or antigen-binding
fragment of embodiment 22, wherein the epitope is in the segment
corresponding to residues D78-N90. [0402] 24. The antibody or
antigen-binding fragment of embodiment 8, wherein the epitope
comprises residues H79 and N90. [0403] 25. The antibody or
antigen-binding fragment of embodiment 24, wherein the epitope
further comprises residue R83. [0404] 26. The antibody or
antigen-binding fragment of embodiment 25, further comprising one
or more of S85, F91, and L92. [0405] 27. The antibody of any of the
preceding embodiments, which is a human or humanized antibody.
[0406] 28. The antibody or antigen-binding fragment of any of the
preceding embodiments, comprising a heavy chain variable region
that is the product of or derived from a set of human genes
comprising VH1.sub.--03, D3-10, and JH6 genes. [0407] 29. The
antibody or antigen-binding fragment of any of the preceding
embodiments, comprising a light-chain variable region that is the
product of or derived from a set of human genes comprising VKI_L18
and JK4 genes. [0408] 30. The antibody or antigen-binding fragment
of any of the preceding embodiments, which is a full-length
antibody. [0409] 31. The antibody of embodiment 30, which is a
human antibody of the IgG1, IgG2, or IgG3 isotype. [0410] 32. The
antibody of embodiment 30, which is of the IgG4 isotype. [0411] 33.
The antibody of embodiment 32, which comprises an S241P mutation.
[0412] 34. An antibody derivative or multispecific antibody
molecule comprising the antibody or antigen-binding fragment of any
of embodiments 1-33. [0413] 35. An isolated anti-hIL20 antibody or
an antigen-binding fragment thereof, comprising the heavy-chain
variable regions CDR2 and CDR3 of SEQ ID NO:8. [0414] 36. The
antibody or antigen-binding fragment of embodiment 35, comprising
the heavy-chain variable region CDR1 of SEQ ID NO:8. [0415] 37. The
antibody or antigen-binding fragment of embodiment 36, comprising a
heavy-chain variable region comprising the sequence of SEQ ID NO:8.
[0416] 38. The antibody or antigen-binding fragment of embodiment
35, comprising the heavy-chain variable region CDR2 and CDR3 of SEQ
ID NO:6. [0417] 39. The antibody or antigen-binding fragment of
embodiment 38, comprising the heavy-chain variable region CDR1 of
SEQ ID NO:6. [0418] 40. The antibody or antigen-binding fragment of
embodiment 39, comprising a heavy-chain variable region comprising
the sequence of SEQ ID NO:6. [0419] 41. The antibody or
antigen-binding fragment of embodiment 35, comprising the
heavy-chain variable region CDR2 and CDR3 of SEQ ID NO:7. [0420]
42. The antibody or antigen-binding fragment of embodiment 41,
comprising the heavy-chain variable region CDR1 of SEQ ID NO:7.
[0421] 43. The antibody or antigen-binding fragment of embodiment
42, comprising a heavy-chain variable region comprising the
sequence of SEQ ID NO:7. [0422] 44. The antibody or antigen-binding
fragment of any of embodiments 35-43, comprising the light-chain
variable region CDR1, CDR2 and CDR3 of SEQ ID NO:9. [0423] 45. The
antibody or antigen-binding fragment of embodiment 44, comprising a
light-chain variable region comprising the sequence of SEQ ID NO:9.
[0424] 46. An isolated human anti-IL20 antibody which competes with
an antibody comprising a VH region comprising SEQ ID NO:6 and/or 7
and a VL region comprising SEQ ID NO:9 in binding to mature human
IL20 (SEQ ID NO:1). [0425] 47. The antibody of embodiment 46, which
further competes with an antibody comprising a VH region comprising
SEQ ID NO:6 and/or 7 and a VL region comprising SEQ ID NO:9 in
binding to mIL20 (SEQ ID NO:4), cIL20 (SEQ ID NO:5), or both.
[0426] 48. The antibody of embodiment 46, which binds to an epitope
comprising at least one residue in the segment corresponding to
residues D78-H103 of mature human IL20 (SEQ ID NO:1), optionally
excluding D78. [0427] 49. The antibody of embodiment 48, which
binds to an epitope comprising residue H79, R83, S85, N90, F91
and/or L92. [0428] 50. The antibody of embodiment 46, which binds
to the same epitope as an antibody comprising a VH region
comprising SEQ ID NO:6 and/or 7 and a VL region comprising SEQ ID
NO:9 in hIL20 (SEQ ID NO:1). [0429] 51. An antigen-binding fragment
of the antibody of any of embodiments 46-50. [0430] 52. A method of
producing an anti-IL20 antibody or antigen-binding fragment,
comprising culturing a host cell producing the antibody or
antigen-binding fragment of any of the preceding embodiments under
suitable conditions, and recovering said antibody or
antigen-binding fragment. [0431] 53. A composition comprising the
antibody or antigen-binding fragment of any of the preceding
embodiments, and a pharmaceutically acceptable carrier. [0432] 54.
The composition of embodiment 53, further comprising a second
anti-inflammatory agent. [0433] 55. The composition of embodiment
54, wherein the second anti-inflammatory agent is selected from an
immunosuppressant, an analgesic, an anti-angiogenic agent, a
corticosteroid, a B-cell depletion agent, a B-cell antagonist, a
T-cell antagonist, a complement-inhibiting agent, an anti-cytokine
agent, and an anti-cytokine receptor agent, and combinations
thereof. [0434] 56. A method for treating an inflammatory or
autoimmune disorder, comprising administering an effective amount
of an anti-IL20 antibody or an antigen-binding fragment thereof,
which antibody or antigen-binding fragment reduces human IL20
mediated activation of human IL20R1/hIL20R2 and IL22R1/hIL20R2
receptor complexes. [0435] 57. The method of embodiment 56,
comprising administering a second anti-inflammatory agent before,
simultaneously with, or after administration of the composition
comprising the antibody or antigen-binding fragment. [0436] 58. The
method of embodiment 57, wherein the second anti-inflammatory agent
is selected from an immunosuppressant, an analgesic, an
anti-angiogenic agent, a corticosteroid, a B-cell depletion agent,
a B-cell antagonist, a T-cell antagonist, a complement-inhibiting
agent, an anti-cytokine agent, and an anti-cytokine receptor agent,
and combinations thereof. [0437] 59. The method of embodiment 58,
wherein the second anti-inflammatory agent is methotrexate. [0438]
60. The method of any of embodiments 56-59, wherein the
inflammatory or autoimmune disorder is rheumatoid arthritis,
juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis,
ankylosing spondylitis, Sjogren's syndrome, multiple sclerosis,
inflammatory bowel disease, systemic lupus erythematosus, lupus
nephritis, or a combination of any thereof. [0439] 61. The method
of embodiment 60, wherein the inflammatory or autoimmune disorder
is rheumatoid arthritis. [0440] 62. The method of embodiment 60,
wherein the inflammatory or autoimmune disorder is psoriasis.
[0441] 63. The method of embodiment 60, wherein the inflammatory or
autoimmune disorder is psoriatic arthritis. [0442] 64. The method
of embodiment 60, wherein the inflammatory or autoimmune disorder
is multiple sclerosis. [0443] 65. The method of embodiment 60,
wherein the inflammatory or autoimmune disorder is inflammatory
bowel disease. [0444] 66. The method of embodiment 60, wherein the
inflammatory or autoimmune disorder is systemic lupus
erythematosus. [0445] 67. The method of embodiment 60, wherein the
inflammatory or autoimmune disorder is lupus nephritis. [0446] 68.
The antibody or antigen-binding fragment of any of embodiments 1-51
for use in treating an inflammatory or autoimmune disorder. [0447]
69. A combination of the antibody or antigen-binding fragment of
any of embodiments 1-51 with a second anti-inflammatory agent for
use in treating an inflammatory or autoimmune disorder. [0448] 70.
The combination of embodiment 69, wherein the second
anti-inflammatory agent is administered before, simultaneously
with, or after the antibody or antigen-binding fragment. [0449] 71.
The combination of embodiment 70, wherein the second
anti-inflammatory agent is methotrexate. [0450] 72. The use of the
antibody or antigen-binding fragment of any of embodiments 1-51 in
the preparation of a medicament for treating an inflammatory or
autoimmune disorder. [0451] 73. The antibody or antigen-binding
fragment, combination, or use of any of embodiments 68-72, wherein
the inflammatory or autoimmune disorder is rheumatoid arthritis.
[0452] 74. The antibody or antigen-binding fragment, combination,
or use of any of embodiments 68-72, wherein the inflammatory or
autoimmune disorder is psoriasis. [0453] 75. The antibody or
antigen-binding fragment, combination, or use of any of embodiments
68-72, wherein the inflammatory or autoimmune disorder is psoriatic
arthritis. [0454] 76. The antibody or antigen-binding fragment,
combination, or use of any of embodiments 68-72, wherein the
inflammatory or autoimmune disorder is multiple sclerosis. [0455]
77. The antibody or antigen-binding fragment, combination, or use
of any of embodiments 68-72, wherein the inflammatory or autoimmune
disorder is inflammatory bowel disease. [0456] 78. The antibody or
antigen-binding fragment, combination, or use of any of embodiments
68-72, wherein the inflammatory or autoimmune disorder is systemic
lupus erythematosus. [0457] 79. The antibody or antigen-binding
fragment, combination, or use of any of embodiments 68-72, wherein
the inflammatory or autoimmune disorder is lupus nephritis. [0458]
80. The antibody or antigen-binding fragment, combination, or use
of any of embodiments 68-72, wherein the inflammatory or autoimmune
disorder is juvenile rheumatoid arthritis. [0459] 81. The antibody
or antigen-binding fragment, combination, or use of any of
embodiments 68-72, wherein the inflammatory or autoimmune disorder
is ankylosing spondylitis. [0460] 82. The antibody or
antigen-binding fragment, combination, or use of any of embodiments
68-72, wherein the inflammatory or autoimmune disorder is Sjogren's
syndrome.
Sequence CWU 1
1
461152PRTHomo sapiens 1Leu Lys Thr Leu Asn Leu Gly Ser Cys Val Ile
Ala Thr Asn Leu Gln 1 5 10 15 Glu Ile Arg Asn Gly Phe Ser Glu Ile
Arg Gly Ser Val Gln Ala Lys 20 25 30 Asp Gly Asn Ile Asp Ile Arg
Ile Leu Arg Arg Thr Glu Ser Leu Gln 35 40 45 Asp Thr Lys Pro Ala
Asn Arg Cys Cys Leu Leu Arg His Leu Leu Arg 50 55 60 Leu Tyr Leu
Asp Arg Val Phe Lys Asn Tyr Gln Thr Pro Asp His Tyr 65 70 75 80 Thr
Leu Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe Leu Thr Ile Lys 85 90
95 Lys Asp Leu Arg Leu Cys His Ala His Met Thr Cys His Cys Gly Glu
100 105 110 Glu Ala Met Lys Lys Tyr Ser Gln Ile Leu Ser His Phe Glu
Lys Leu 115 120 125 Glu Pro Gln Ala Ala Val Val Lys Ala Leu Gly Glu
Leu Asp Ile Leu 130 135 140 Leu Gln Trp Met Glu Glu Thr Glu 145 150
2176PRTHomo sapiens 2Met Lys Ala Ser Ser Leu Ala Phe Ser Leu Leu
Ser Ala Ala Phe Tyr 1 5 10 15 Leu Leu Trp Thr Pro Ser Thr Gly Leu
Lys Thr Leu Asn Leu Gly Ser 20 25 30 Cys Val Ile Ala Thr Asn Leu
Gln Glu Ile Arg Asn Gly Phe Ser Glu 35 40 45 Ile Arg Gly Ser Val
Gln Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile 50 55 60 Leu Arg Arg
Thr Glu Ser Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys 65 70 75 80 Cys
Leu Leu Arg His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys 85 90
95 Asn Tyr Gln Thr Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu
100 105 110 Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys
His Ala 115 120 125 His Met Thr Cys His Cys Gly Glu Glu Ala Met Lys
Lys Tyr Ser Gln 130 135 140 Ile Leu Ser His Phe Glu Lys Leu Glu Pro
Gln Ala Ala Val Val Lys 145 150 155 160 Ala Leu Gly Glu Leu Asp Ile
Leu Leu Gln Trp Met Glu Glu Thr Glu 165 170 175 3177PRTHomo sapiens
3Met Lys Leu Gln Cys Val Ser Leu Trp Leu Leu Gly Thr Ile Leu Ile 1
5 10 15 Leu Cys Ser Val Asp Asn His Gly Leu Arg Arg Cys Leu Ile Ser
Thr 20 25 30 Asp Met His His Ile Glu Glu Ser Phe Gln Glu Ile Lys
Arg Ala Ile 35 40 45 Gln Ala Lys Asp Thr Phe Pro Asn Val Thr Ile
Leu Ser Thr Leu Glu 50 55 60 Thr Leu Gln Ile Ile Lys Pro Leu Asp
Val Cys Cys Val Thr Lys Asn 65 70 75 80 Leu Leu Ala Phe Tyr Val Asp
Arg Val Phe Lys Asp His Gln Glu Pro 85 90 95 Asn Pro Lys Ile Leu
Arg Lys Ile Ser Ser Ile Ala Asn Ser Phe Leu 100 105 110 Tyr Met Gln
Lys Thr Leu Arg Gln Cys Gln Glu Gln Arg Gln Cys His 115 120 125 Cys
Arg Gln Glu Ala Thr Asn Ala Thr Arg Val Ile His Asp Asn Tyr 130 135
140 Asp Gln Leu Glu Val His Ala Ala Ala Ile Lys Ser Leu Gly Glu Leu
145 150 155 160 Asp Val Phe Leu Ala Trp Ile Asn Lys Asn His Glu Val
Met Phe Ser 165 170 175 Ala 4176PRTMus musculus 4Met Lys Gly Phe
Gly Leu Ala Phe Gly Leu Phe Ser Ala Val Gly Phe 1 5 10 15 Leu Leu
Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His Leu Gly Ser 20 25 30
Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu 35
40 45 Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp Ile Arg
Ile 50 55 60 Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys Ser Leu
Asp Arg Cys 65 70 75 80 Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu
Asp Arg Val Phe Lys 85 90 95 Val Tyr Gln Thr Pro Asp His His Thr
Leu Arg Lys Ile Ser Ser Leu 100 105 110 Ala Asn Ser Phe Leu Ile Ile
Lys Lys Asp Leu Ser Val Cys His Ser 115 120 125 His Met Ala Cys His
Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln 130 135 140 Ile Leu Ser
His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys 145 150 155 160
Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu 165
170 175 5176PRTMacaca fascicularis 5Met Lys Ala Ser Ser Leu Ala Phe
Ser Leu Leu Ser Ala Ala Phe Tyr 1 5 10 15 Leu Leu Trp Thr Pro Ser
Thr Gly Leu Lys Thr Leu Asn Leu Gly Ser 20 25 30 Cys Val Ile Ala
Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe Ser Glu 35 40 45 Ile Arg
Gly Ser Val Gln Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile 50 55 60
Leu Arg Arg Thr Glu Ser Leu Gln Asp Thr Lys Pro Ala Asp Gln Cys 65
70 75 80 Cys Leu Leu Arg His Leu Leu Arg Leu Tyr Leu Asp Arg Val
Phe Lys 85 90 95 Asn Tyr Gln Thr Leu Asp His Tyr Thr Leu Arg Lys
Ile Ser Ser Leu 100 105 110 Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp
Leu Arg Leu Cys His Ala 115 120 125 His Met Thr Cys His Cys Gly Glu
Glu Ala Met Lys Lys Tyr Gly Gln 130 135 140 Ile Leu Ser His Phe Glu
Glu Leu Glu Pro Gln Ala Ala Val Val Lys 145 150 155 160 Ala Leu Gly
Glu Leu Asp Ile Leu Leu Gln Trp Met Glu Glu Thr Glu 165 170 175
6127PRTHomo sapiens 6Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Asp 20 25 30 Ile Ile His Trp Val Arg Gln
Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala
Gly Tyr Gly Asn Thr Gln Tyr Ser Gln Asn Phe 50 55 60 Gln Asp Arg
Val Ser Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ile Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Pro Leu Trp Phe Gly Glu Ser Ser Pro His Asp Tyr Tyr
100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 125 7127PRTHomo sapiens 7Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser His 20 25 30 Ile Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly
Trp Ile Asn Ala Gly Tyr Gly Asn Thr Lys Tyr Ser Gln Asn Phe 50 55
60 Gln Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ile Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Pro Leu Trp Phe Gly Glu Leu Ser Pro
His Asp Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 125 8127PRTHomo
sapiensMISC_FEATURE(30)..(30)X1 is T, S, or a conservative
substitution of any thereof 8Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Xaa Xaa Xaa 20 25 30 Ile Xaa His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile
Asn Ala Gly Tyr Gly Asn Thr Xaa Tyr Ser Gln Asn Phe 50 55 60 Gln
Asp Arg Val Xaa Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ile Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Glu Pro Leu Trp Phe Gly Glu Xaa Ser Pro His
Asp Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120 125 9108PRTHomo sapiens 9Ala Ile Gln Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg 100 105 1098PRTHomo sapiens 10Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met
His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45
Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg 1131DNAHomo sapiens 11gtattactat
ggttcgggga gttattataa c 31129PRTHomo sapiens 12Val Leu Leu Trp Phe
Gly Glu Leu Leu 1 5 1363DNAHomo sapiens 13attactacta ctactacggt
atggacgtct gggggcaagg gaccacggtc accgtctcct 60cag 631420PRTHomo
sapiens 14Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr
Thr Val 1 5 10 15 Thr Val Ser Ser 20 1595PRTHomo sapiens 15Ala Ile
Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Phe Asn Ser Tyr Pro 85 90 95 1638DNAHomo sapiens 16gctcactttc
ggcggaggga ccaaggtgga gatcaaac 381712PRTHomo sapiens 17Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 18123PRThomo sapiens
18Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1
5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Phe Ser Thr
Asn 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys
Arg Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys
Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met
Asp Pro Leu Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Ser Pro
Phe Ile Met Val Arg Gly Val Ile Ile Thr Phe 100 105 110 Phe Asp Phe
Trp Gly Gln Gly Thr Leu Val Thr 115 120 19109PRTHomo sapiens 19Val
Ile Trp Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly 1 5 10
15 Asp Arg Val Thr Ile Ser Cys Arg Met Ser Gln Gly Ile Ser Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu
Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Cys Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Tyr Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg Thr 100 105 20123PRTHomo sapiens 20Gln Leu
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20
25 30 Asn Tyr Tyr Trp Ala Trp Ile Arg Gln Pro Pro Gly Lys Gly Pro
Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr
Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Ala Ala Val Tyr Phe 85 90 95 Cys Ala Gly Leu Val Val Ile
Pro Ala Ser Asp Tyr Ser Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly
Gln Gly Thr Ser Val Thr 115 120 21110PRTHomo sapiens 21Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg Thr 100 105 110 22122PRTHomo sapiens 22Gln Val
Gln Val Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Arg Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20
25 30 Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45 Gly Trp Ile Asn Ala Gly Tyr Gly Asn Thr Lys Tyr Ser
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser
Ala Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Ala Leu
Ser Trp Phe Gly Glu Ser Gln Gly Phe 100 105 110 Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 115 120 23122PRTHomo sapiens 23Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Arg Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe Pro Asn Tyr
20 25 30 Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu
Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Tyr Gly Asn Thr Lys Tyr
Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr
Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Val
Leu Leu Trp Phe Gly Glu Ser Gln Gly Phe 100 105 110 Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr 115 120 24108PRTHomo sapiens 24Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Ser Pro 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr 100 105 25476PRTHomo sapiens 25Met Asp Trp Thr Trp
Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg 20 25 30 Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40
45 Thr Asn Asp Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu
50 55 60 Glu Trp Met Gly Trp Ile Asn Ala Gly Tyr Gly Asn Thr Gln
Tyr Ser 65 70 75 80 Gln Asn Phe Gln Asp Arg Val Ser Ile Thr Arg Asp
Thr Ser Ala Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ile Ser Leu Arg
Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Glu Pro Leu
Trp Phe Gly Glu Ser Ser Pro His 115 120 125 Asp Tyr Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val 130 135 140 Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170
175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295
300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420
425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 465 470 475 26552PRTMacaca fascicularis 26Met Arg Ala Pro Ser
Ser Pro Ala Leu Arg Pro Leu Leu Pro Pro Leu 1 5 10 15 Leu Leu Leu
Leu Leu Ala Ala Pro Trp Gly Leu Ala Val Pro Cys Val 20 25 30 Ser
Gly Gly Leu Pro Lys Pro Ala Asn Ile Thr Phe Leu Ser Ile Asn 35 40
45 Met Lys Asn Val Leu Gln Trp Asn Pro Pro Glu Cys Leu Gln Gly Val
50 55 60 Lys Val Thr Tyr Thr Val Gln Tyr Phe Ile Tyr Gly Gln Lys
Lys Trp 65 70 75 80 Leu Asn Lys Ser Glu Cys Arg Asn Ile Asn Arg Thr
Tyr Cys Asp Leu 85 90 95 Ser Ala Glu Thr Ser Asp Tyr Glu His Gln
Tyr Tyr Ala Lys Val Lys 100 105 110 Ala Ile Trp Gly Thr Asn Cys Ser
Lys Trp Ala Glu Ser Gly Arg Phe 115 120 125 Tyr Pro Phe Leu Glu Thr
Gln Ile Gly Pro Pro Glu Val Ala Leu Thr 130 135 140 Thr Asp Glu Lys
Ser Ile Ser Val Val Leu Thr Ala Pro Glu Lys Trp 145 150 155 160 Lys
Arg Asn Pro Glu Asp Leu Pro Val Ser Met Arg Gln Ile Tyr Ser 165 170
175 Asn Leu Lys Tyr Asn Val Ser Val Ser Asn Thr Lys Ser Asn Arg Thr
180 185 190 Trp Ser Gln Cys Val Thr Asn His Thr Leu Val Leu Thr Trp
Leu Glu 195 200 205 Pro Asn Thr Leu Tyr Cys Ile His Val Glu Ser Phe
Val Pro Gly Pro 210 215 220 Pro Arg Arg Ala Gln Pro Ser Glu Lys Gln
Cys Ala Arg Thr Leu Lys 225 230 235 240 Asp Gln Ser Ser Glu Phe Lys
Ala Lys Ile Ile Phe Trp Tyr Val Leu 245 250 255 Pro Val Ser Val Thr
Val Phe Leu Phe Ser Val Met Gly Tyr Ser Ile 260 265 270 Tyr Arg Tyr
Ile His Val Gly Lys Glu Lys His Pro Ala Asn Leu Ile 275 280 285 Leu
Ile Tyr Gly Asn Glu Phe Asp Lys Arg Phe Phe Val Pro Ala Glu 290 295
300 Lys Ile Val Ile Asn Phe Ile Thr Leu Asn Ile Ser Asp Asp Ser Lys
305 310 315 320 Ile Ser His Gln Asp Met Ser Leu Leu Gly Lys Ser Ser
Asp Val Ser 325 330 335 Ser Leu Asn Asp Pro Gln Pro Ser Gly Asn Leu
Lys Pro Pro Gln Glu 340 345 350 Glu Glu Glu Val Lys His Leu Gly Tyr
Ala Ser His Leu Met Glu Ile 355 360 365 Val Cys Asp Ser Glu Glu Asn
Ala Glu Gly Thr Ser Leu Thr Gln Gln 370 375 380 Ala Ser Leu Ser Arg
Thr Ile Pro Pro Asp Lys Thr Val Ile Glu Tyr 385 390 395 400 Glu Cys
Asp Val Arg Thr Thr Asp Ile Cys Ala Gly Pro Glu Glu Gln 405 410 415
Glu Leu Arg Leu Gln Glu Glu Val Ser Thr Gln Gly Thr Leu Leu Glu 420
425 430 Ser Gln Ala Ala Leu Ala Leu Leu Gly Pro Gln Thr Leu Gln Tyr
Ser 435 440 445 Tyr Thr Pro Gln Leu Gln Asp Leu Asp Pro Leu Thr Arg
Glu His Thr 450 455 460 Asp Ser Glu Glu Gly Pro Glu Glu Glu Pro Ser
Thr Thr Leu Val Asp 465 470 475 480 Trp Asp Pro Gln Thr Gly Arg Leu
Cys Ile Pro Ser Leu Ser Ser Phe 485 490 495 Asp Gln Asp Ser Glu Gly
Cys Glu Pro Ser Glu Gly Asp Gly Leu Gly 500 505 510 Glu Glu Gly Leu
Leu Ser Arg Leu Tyr Glu Glu Pro Ala Pro Asp Arg 515 520 525 Pro Pro
Gly Glu Asn Glu Thr Tyr Leu Met Gln Phe Met Glu Glu Trp 530 535 540
Gly Leu Tyr Val Gln Met Glu Asn 545 550 27311PRTMacaca fascicularis
27Met Gln Thr Phe Thr Met Val Leu Gln Glu Ile Trp Thr Ser Leu Phe 1
5 10 15 Met Trp Phe Phe Tyr Ala Leu Ile Pro Cys Leu Leu Thr Asp Glu
Val 20 25 30 Ala Ile Leu Pro Ala Pro Gln Asn Leu Ser Val Leu Ser
Thr Asn Met 35 40 45 Lys His Leu Leu Met Trp Ser Pro Val Thr Val
Pro Gly Glu Thr Val 50 55 60 Tyr Tyr Ser Val Glu Tyr Gln Gly Glu
Tyr Glu Ser Leu Tyr Thr Ser 65 70 75 80 His Ile Trp Ile Pro Ser Ser
Trp Cys Ser Leu Thr Glu Gly Pro Glu 85 90 95 Cys Asp Val Thr Asp
Asp Ile Thr Ala Thr Val Pro Tyr Asn Leu Arg 100 105 110 Val Arg Ala
Thr Leu Gly Ser Gln Thr Ser Ala Trp Ser Ile Leu Lys 115 120 125 His
Pro Phe Asn Arg Asn Ser Thr Ile Leu Thr Pro Pro Gly Met Glu 130 135
140 Ile Thr Lys Asp Gly Phe His Leu Val Ile Glu Leu Glu Asp Leu Gly
145 150 155 160 Pro Gln Phe Glu Phe Leu Val Ala Tyr Trp Arg Arg Glu
Pro Gly Ala 165 170 175 Glu Glu His Val Lys Met Val Arg Ser Gly Gly
Ile Pro Val His Leu 180 185 190 Glu Thr Met Glu Pro Gly Ala Ala Tyr
Cys Val Lys Ala Gln Thr Phe 195 200 205 Val Lys Ala Ile Gly Arg Tyr
Ser Ala Phe Ser Gln Thr Glu Cys Val 210 215 220 Glu Val Gln Gly Glu
Ala Ile Pro Leu Val Leu Ala Leu Phe Ala Phe 225 230 235 240 Val Gly
Phe Met Leu Ile Leu Val Val Val Pro Leu Phe Val Trp Lys 245 250 255
Met Gly Arg Leu Leu Gln Tyr Ser Cys Cys Pro Val Val Val Leu Pro 260
265 270 Asp Thr Leu Lys Ile Thr Asn Ser Pro Gln Lys Leu Ile Ser Cys
Arg 275 280 285 Arg Glu Glu Val Asp Ala Cys Ala Thr Ala Val Met Ser
Pro Glu Glu 290 295 300 Leu Leu Arg Ala Trp Ile Ser 305 310
28574PRTMacaca fascicularis 28Met Arg Thr Leu Leu Thr Ile Leu Ala
Val Gly Ser Leu Ala Ala His 1 5 10 15 Ala Pro Glu Asp Pro Ser Asp
Leu Leu Gln His Val Lys Phe Gln Ser 20 25 30 Asn Asn Phe Glu Asn
Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr 35 40 45 Pro Asp Thr
Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp 50 55 60 Trp
Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn 65 70
75 80 Leu Thr Val Glu Thr Gly Asn His Thr Glu Leu Tyr Tyr Ala Arg
Val 85 90 95 Thr Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met
Thr Asp Arg 100 105 110 Phe Asn Ser Leu Gln His Thr Ala Leu Lys Pro
Pro Asp Val Thr Cys 115 120 125 Ile Pro Lys Val Arg Ser Ile Gln Met
Ile Val His Pro Thr Pro Thr 130 135 140 Pro Ile Arg Ala Gly Asp Gly
His Arg Leu Thr Leu Glu Asp Ile Phe 145 150 155 160 His Asp Leu Phe
Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln 165 170 175 Met His
Leu Gly Gly Glu Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr 180 185 190
Pro Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro Thr Trp 195
200 205 Ser Lys Lys Ser Ala Pro Tyr Met Cys Arg Val Arg Thr Leu Pro
Asp 210 215 220 Arg Thr Trp Thr Tyr Ser Phe Ser Gly Ala Phe Leu Phe
Ser Met Gly 225 230 235 240 Phe Leu Val Ala Val Leu Cys Tyr Leu Ser
Tyr Arg Tyr Val Thr Lys 245 250 255 Pro Pro Ala Pro Pro Asn Ser Leu
Asn Val Gln Arg Val Leu Thr Phe 260 265 270 Gln Pro Leu Arg Phe Ile
Gln Glu His Val Leu Ile Pro Ala Phe Asp 275 280 285 Leu Ser Gly Pro
Ser Ser Leu Ala Gln Pro Val Gln Tyr Ser Gln Ile 290 295 300 Arg Val
Ser Gly Pro Arg Glu Pro Ala Gly Pro Pro Gln Arg His Ser 305 310 315
320 Leu Ser Glu Ile Thr Tyr Leu Gly Gln Pro Asp Ile Ser Ile Leu Gln
325 330 335 Pro Ala Asn Val Pro Pro Pro Gln Ile Leu Ser Pro Leu Ser
Tyr Ala 340 345 350 Pro Asn Ala Ala Pro Glu Val Gly Pro Pro Ser Tyr
Ala Pro Gln Val 355 360 365 Thr Pro Glu Ala Gln Leu Pro Phe Tyr Thr
Pro Gln Ala Val Ser Lys 370 375 380 Val Gln Pro Pro Ser Tyr Ala Pro
Gln Ala Thr Pro Asp Ser Trp Pro 385 390 395 400 Pro Ser Tyr Gly Val
Cys Val Glu Gly Ser Gly Lys Asp Ser Pro Thr 405 410 415 Val Thr Leu
Ser Ser Pro Lys His Leu Arg Pro Lys Gly Gln Leu Gln 420 425 430 Lys
Glu Pro Pro Ala Gly Ser Cys Met Ser Gly Gly Leu Ser Leu Gln 435 440
445 Glu Val Thr Ser Leu Ala Met Glu Glu Ser Gln Glu Ala Lys Ser Leu
450 455 460 His Gln Pro Leu Gly Val Cys Thr Asp Arg Thr Ser Asp Leu
Asn Val 465 470 475 480 Leu Asp Ser Gly Glu Glu Gly Thr Pro Gln Tyr
Leu Lys Gly Gln Leu 485 490 495 Pro Leu Leu Ser Ser Val Gln Ile Glu
Gly His Pro Met Ser Leu Pro 500 505 510 Leu His Pro Pro Ser Arg Pro
Cys Ser Pro Ser Asp Gln Gly Pro Ser 515 520 525 Pro Trp Gly Leu Leu
Glu Ser Leu Val Cys Pro Lys Asp Glu Ala Lys 530 535 540 Ser Leu Ala
Pro Glu Thr Ser Asp Leu Glu Gln Pro Thr Glu Leu Asp 545 550 555 560
Ser Leu Phe Arg Gly Leu Ala Leu Thr Val Gln Trp Glu Ser 565 570
2930DNAHomo sapiens 29agaattccac catgaggacg ctgctgacca
303026DNAHomo sapiens 30gctcgagaca gggaggaagc accaag 263125DNAHomo
sapiens 31cgaattccct tggtttctgg ggaag 253227DNAHomo sapiens
32gctcgagcac aggaaacaaa aggcaaa 273333DNAHomo sapiens 33agaattctgg
aaagaaacaa tgttctaggt caa 333425DNAHomo sapiens 34gctcgagctt
cacctgggcc cttcc 253535DNAMus musculus 35ccgaattcgc caccatgaag
acactactga ccatc 353633DNAMus musculus 36cttgcggccg ctcaggattc
ccactgcaca gtc 333730DNAMus musculus 37ttgaattcgc caccatgcac
actcccggga 303836DNAMus musculus 38ttgcggccgc ctagctttcc atttgtacat
gtaacc 363934DNAMus musculus 39ttggatccgc caccatgatt tcccagggag
tctg 344031DNAMus musculus 40ttgcggccgc tcaagtctgt gagatccaga c
314130DNAMacaca fascicularis 41agaattccac catgaggacg ctgctgacca
304226DNAMacaca fascicularis 42gctcgagaca gggaggaagc accaag
264321DNAMacaca fascicularis 43gtgggactga gcagtctgct g
214421DNAMacaca fascicularis 44aggcaaaagg aagtgttggc a
214533DNAMacaca fascicularis 45agaattctgg aaagaaacaa tgttctaggt caa
334625DNAMacaca fascicularis 46gctcgagctt cacctgggcc cttcc 25
* * * * *
References