Note: Descriptions are shown in the official language in which they were submitted.
WO 2012/021773 CA 02808185 2013-02-12PCT/US2011/047532
ANTIBODIES TO IL-113 AND IL-18, FOR TREATMENT OF DISEASE
RELATED APPLICATION
[0001] This non-provisional application claims the benefit of priority of U.S.
Provisional
Application Serial No. 61/373,760 filed 13 August 2010, which is incorporated
by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to anti-IL-113 and anti-IL-18
antibodies,
including anti-IL-113 and anti-IL-18 bispecific antibodies and monoclonal
antibodies, and
methods of using such antibodies for the treatment of disease.
BACKGROUND
[0003] The interleukin-1 (1L-1) and IL-18 family of cytokines are related by
mechanism of
origin, receptor structure, and signal transduction pathways utilized. These
cytokines are
synthesized as precursor molecules and cleaved by the enzyme caspase-1 before
or during
release from the cell. The NALP-3 inflammasome is of crucial importance in
generating active
caspase-1 (Cassel et al., 2009; Ferrero-Miliani et al., 2007). The IL-1 family
contains two
agonists, 1L-1a and IL-1[3, a specific inhibitor, IL-1 receptor antagonist (1L-
1Ra), and two
receptors, the biologically active type IL-1R and inactive type II IL-1R
(Arend et al., 2008).
Both IL-1R1 and IL-33R utilize the same interacting accessory protein (1L-
1RAcP). The
balance between IL-1 and IL-1Ra is important in preventing disease in various
organs, and
excess production of 1L-1 has been implicated in many human diseases. The IL-
18 family
also contains a specific inhibitor, the IL-18-binding protein (1L-18BP), which
binds IL-18 in the
fluid phase. The IL-18 receptor is similar to the IL-1 receptor complex,
including a single
ligand-binding chain and a different interacting accessory protein. IL-18
provides an
important link between the innate and adaptive immune responses.
[0004] Inflammasome activation and IL-1[3/1L-18 processing and secretion may
be
involved in disease progression. Genome-wide association studies indicate a
role for the
inflammasome in inflammatory bowel disease (IBD). Patients with polymorphisms
in the
inflammasome-compound NALP-3 are reportedly at increased risk for Crohn's
disease
(Ferrero-Miliani et al., 2007; Villani et al., 2009). In addition,
polymorphisms in autophagy
components Atg16I1 and IRGM that control caspase-1 activation and IL-113/1L-18
processing
have been reportedly linked to Crohn's disease (Baldassano et al., 2007;
Cadwell et al., 2008;
Kuballa et al., 2008; Saitoh et al., 2008). Independent studies have reported
increased serum
WO 2012/021773 CA 02808185 2013-02-12 PCT/US2011/047532
levels of IL-113 and IL-18 in patients with IBD (Ludwiczek et al., 2005;
Ludwiczek et al., 2004;
Monteleone et al., 1999). Studies in humans have been further supported by
preclinical
studies. Blockade of IL-18 or IL-113 reportedly leads to amelioration of
clinical scores in
preclinical models of the disease (Ten Hove et al., 2001).
[0005] Further, it has been reported that in the eye, there are increased
levels of IL-113 in
patients with diabetic retinopathy (Kowluru and Odenbach, 2004).
SUMMARY OF THE INVENTION
[0006] The present invention provides anti-IL-1[3 and anti-IL-18 antibodies,
including e.g.,
anti-IL-113 and anti-IL-18 bispecific antibodies, and methods of using such
antibodies for
treatment of disease. In some embodiments, the anti-IL-113 and anti-IL-18
antibodies are
monoclonal antibodies, and are administered concurrently or consecutively to a
patient, for
treatment of disease. In other embodiments, the anti-IL-113 and anti-IL-18 are
bispecific
antibodies and are administered to a patient for treatment of disease. In some
embodiments
the disease is an inflammasome-mediated disease, e.g., a disease wherein the
inflammasome is activated. Examples of diseases include immune diseases and
autoimmune diseases, and include inflammatory bowel disease (IBD), age-related
macular
degeneration (AMD), and type 2 diabetes (T2D).
[0007] In some embodiments, the anti-IL-113 and anti-IL-18 antibodies of the
present
invention, block or neutralize the activity of, and/or bind to, IL-113 and/or
IL-18. In some
embodiments the bispecific antibody blocks or neutralizes the activity or,
and/or binds to, IL-
113 and/or IL-18.
[0008] In one aspect, there is provided a method of treating a disease in a
patient, the
method comprising administering to said patient an effective amount of:
a. An I L-18/IL-18 bispecific antibody; or
b. An antibody that binds IL-1 13 and IL-18; or
c. An antibody that binds IL-1 13 and an antibody that binds IL-18;
wherein said antibody or antibodies of parts a, b or c is/are capable of
neutralizing or blocking
IL-113 and IL-18 activity in cells or tissue.
[0009] In some embodiments, the antibody/antibodies used in the method is/are
humanized. In some embodiments, the antibody is a dual action antibody.
[0010] In some embodiments, the method uses a combined treatment comprising
an
anti-IL-113 antibody and an anti-IL-18 antibody. In one embodiment, at least
one antibody is
monoclonal. In some embodiments, each antibody is monoclonal. In some
embodiments,
the antibodies of part (c) are given simultaneously, or consecutively. In some
embodiments,
the antibodies are administered within one hour.
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[0011] In some embodiments, the disease to be treated is an immune disease or
an
autoimmune disease or an inflammatory or an autoinflammatory disease. In some
embodiments, the disease is an inflammasome-mediated disease. In some
embodiments,
the disease is an IL-1[3 related disease or an IL-18 related disease or an IL-
1[3/IL-18 13
disease.
[0012] In some embodiments, the disease is age-related macular degeneration
(AMD). In
some embodiments, the disease is type 2 diabetes (T2D). In some embodiments,
the
inflammatory bowel disease (IBD). In some embodiments, the disease is Crohn's
disease
(CD). In some embodiments, the disease is ulcerative colitis (UC). In some
embodiments, the
disease is atherosclerosis. In some embodiments, the disease is cardio-
metabolic disease. In
some embodiments, the disease is fibrostenosing Crohn's disease.
[0013] In some embodiments, the patient being treated by the method has not
responded
to anti-TNF therapy.
[0014] In some embodiments, the method of treating disease in a patient
comprises
administering to said patient an effective amount of a monoclonal antibody
that binds IL-113
and a monoclonal antibody that binds IL-18.
[0015] In another aspect, there is provided a method of neutralizing or
blocking IL-113
and/or IL-18 activity in cells or tissue, the method comprising contacting
said cells or tissue
with a monoclonal antibody that binds IL-113 and a monoclonal antibody that
binds IL-18, and
thereby neutralizing or blocking said activity. In some embodiments, the
antibodies are
administered concurrently or consecutively. In some embodiments, the cells are
contacted
concurrently or consecutively with said monoclonal antibody that binds IL-113
and said
monoclonal antibody that binds IL-18.
[0016] In another aspect, there is provided an antibody that neutralizes or
blocks IL-1[3
and IL-18 activity. In some embodiments, the antibody is a bispecific
antibody. In some
embodiments, the antibody is humanized. In some embodiments, the antibody
binds to IL-113
and IL-18.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 depicts examples of ligands and receptors for IL-113 and for
IL-18. For
example, signaling can be initiated though engagement of two receptor chains
by IL-113 or IL-
18. It is thought that intracellular Toll-Interleukin Rexeptor-like (TIR)
domain leads to
activation of transcription factors NF-kB and AP1 that in turn increase
cytokine production
ultimately resulting in protective immunity, autoinflammatory disorders or
chronic
inflammation.
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[0018] Figure 2 depicts an example of a hypothetical model for IL-18/1L-18
involvement
in inflammatory bowel disease. Stimulation of lamina propria macrophages by
intestinal
microbes leads to autocatalytic activation of caspase-1 that in turn processes
and secretes !L-
IB and IL-18. IL-113 and IL-18 act on various immune cells and induce pro-
inflammatory
cytokines in macrophages, polarize T-cells towards Th1 and Th17 pathogenic T
cells and
disrupt the epithelial barrier, enabling more pathogens to stimulate
macrophages.
[0019] Figure 3 depicts an example of how genetics suggest a role for
inflammasome
activation in Crohn's disease. Polymophisms in autophagy-related genes ATG16L1
and
IRGM and inflammasome regulating genes NOD2 and NALP3 results in increased
Caspase-1
activation and secretion of IL-113 and IL-18.
[0020] Figure 4(A) is data showing that expression of IL-113 and IL-18 mRNA is
increased in colon biopsies from Crohn's and UC patients. The values are based
on relative
intensities of the hybridization signal on an Agilent gene platform. (B) is
data showing that IL-
113 and IL-18 are increased in serum from patients with Crohn's disease and
UC.
[0021] Figure 5 is data showing that differential expression of IL-113 and IL-
18 in inflamed
colon. lmmunohistochemistry on cross-sections through colon biopsies from
patients with UC.
Sections were stained with antibody to human IL-113 and IL-18. While IL-113 is
primarily found
in macrophages present at sites of transmural inflammation, IL-18 is
predominantly found in
dendritic cells present in lymphoid follicles. In both cases, staining was
only observed in
regions of inflammation.
[0022] Figure 6 is data showing that increased secretion of IL-113 and IL-18
from colons
of mice receiving 3.5 % DSS in their drinking water ad libitum for 5 days
[0023] Figure 7 is data showing that increased secretion of IL-113 and IL-18
from colons
of mice receiving adoptively transferred CD4+CD45RBhi T cells.
[0024] Figure 8 is data showing that increased secretion of IL-113 and IL-18
from colons
of IL-10 KO mice treated with piroxicam.
[0025] Figure 9 is data showing that that IL-1R1 and ASC KO mice show
significantly
reduced severity of DSS-induced colitis. Colon scores from mice deficient in
IL-1R1, IL-18Ra
and ASC.
[0026] Figure 10 IL-1R1 deficiency leads to a significant reduction of IL-113,
IL-18, IL-17
and TNF-alpha in DSS-induced colitis.
[0027] Figure 11 is data showing that IL-18R deficiency leads to a significant
reduction in
the levels of IL-18 and IL-12p40 in DSS-induced colitis.
[0028] Figure 12 is data showing that ASC deficiency leads to a significant
reduction in
the levels of IL-18, IL-18, IL-12p40 and IL-17 in DSS-induced colitis.
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[0029] Figure 13 is a summary of exemplary cytokine responses in ex-vivo colon
cultures obtained from various mouse IBD models.
[0030] Figure 14 is data showing that that IL-113 is expressed in vitreous of
a
subpopulation of AMD patients. Vitreous was collected from patients diagnosed
with wet
AMD, geographic atrophy (GA) or from patients with a macular pucker or macular
hole.
Cytokine levels were determined using and ELISA assay.
[0031] Figure 15 is data showing increased IL-113 and Caspase-1 expression in
the eye
following constant light exposure. In (A) Mice were exposed to constant light
(1800 Lux) for
days, after which eyes were removed; in (B) mRNA was isolated from the retina,
and IL-113
mRNA levels were determined by real-time PCR; and in (C) Whole eyes were
homogenized
in lysis buffer, and cell extracts were separated on an SDS gel, blotted and
stained with an
antibody to murine caspase-1.
[0032] Figure 16 is data showing expression of pro-IL-13 and caspase-1 in IL-
18-
infected eyes. Adeno-associated virus (AAV) expressing mature murine IL-113
was injected
sub-retinally. Three weeks later, mice were exposed to intense light (5000
Lux) (ILE; intense
light exposure) for 6 hrs. Eyes were processed 1 day later for Western blot
analysis of IL-113
and Caspase-1 as described in Figure 15.
[0033] Figure 17 is data showing increased inflammation and neo-angiogenesis
following 11_1[3 over-expression in the mouse eye. In (A), albino mice
received a sub-retinal
injection of empty AAV virus or virus expressing IL-113. Three weeks later,
mice were injected
with FITC solution and their eyes were scanned by fluorescein angiography.
Arrow points to
an area with choroidal neovascularization (CNV). In (B), the eyes were
enucleated, fixed and
processed for paraffin embedding and sectioning. Sections were stained with an
antibody to
CD45 to visualize infiltrating immune cells (see inset). Inflammation was
absent in mice sub-
retinally injected with an empty AAV vector.
[0034] Figure 18 is data showing AAV eyes infected with pro-IL-13 show
inflammation
independent of caspase-1 activity. Caspase-1 wt or ko mice were injected
subretinally with
AAV-pro-IL-13 as described for Figure 17. Three weeks later, the eyes were
enucleated and
processed as described. Inflammation proceeded independent of caspase-1
activity.
[0035] Figure 19 is data showing that both AAV-IL113 and AAV-IL-18
significantly reduce
scotopic ERG responses. Electro Retino Grams (ERGs) of mice treated with AAV-
IL-113 and
AAV-1L18 show significant reduction in "a" and "b"-wave responses compared to
mice injected
with empty vector.
[0036] Figure 20 is a summary of the biology of IL-113 and IL-18 in AMD useful
for
developing anti-IL-113 and anti-IL-18 neutralizing antibodies for use in
preclinical studies in
nonhuman animals (e.g., mice), and as a clinical reagent for human studies.
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[0037] Figure 21 is data showing an example of a method for screening of anti-
IL-1[3
neutralizing antibodies using an ELISA-based approach.
[0038] Figure 22 is an ELISA assay showing the neutralizing activity of a
subset of
hamster anti-mouse IL-1[3 hybridomas.
[0039] Figure 23 is a Table illustrating the I050 values for the blocking
activity of
hamster anti-mouse anti-IL-1[3 antibodies.
[0040] Figure 24 is data showing cell lines used to determine neutralizing
activity of
human and murine IL-16/IL-18.
[0041] Figure 25 is data showing (A) the dose-response of NF-kB reporter
activity in an
NIH3T3 cell line treated with increasing concentrations of murine IL-16; and
(B) blocking
activity of hybridoma supernatants containing IL-1[3-neutralizing Abs.
[0042] Figure 26 is a summary of exemplary antibodies derived by phage
technology.
Various phage display libraries with diversity in the heavy chain variable
region (VH) or both the
heavy and light chain variable regions (VHVL) were screened. Also screened was
a synthetic
library (YSGX) which is a reduced genetic codon library which generates
randomized CDRs using
codons enriched in tyrosines, serines and glycines (Fe!louse et al., J Mol
Biol, 373, 924-940) and
a peptide library which is an antibody library designed to potentially bind
specific peptide
sequences.
[0043] Figure 27 is data showing the locking activity of various phage
antibodies in an
ELISA-based neutralization assay.
[0044] Figure 28 is a cartoon depicting an exemplary sequence of events
leading up to
pancreatic beta cell loss and the potential level of intervention with anti-IL-
16/1L-18
neutralizing antibodies.
[0045] Figure 29 is a schematic of the experimental protocol used in Example
4.
[0046] Figure 30 is a graphic displaying the (A) histology colon score results
and (B)
visual colon score results of anti-IL-18 and/or anti-IL18 treatment in the
piroxicam IL-10K0
mouse IBD model. Also shown is the result of TNF-alpha blockade. The anti-IL-
18 and anti-
I L18 combination treatment was equally effective as TNF blockade.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention provides anti-IL-113 and anti-IL-18 antibodies,
including e.g.,
anti-IL-113 and anti-IL-18 bispecific antibodies, and methods of using such
antibodies for
treatment of disease. In some embodiments, the anti-IL-113 and anti-IL-18
antibodies are
monoclonal antibodies, and are administered concurrently or consecutively to a
patient, for
treatment of disease. In other embodiments, the anti-IL-113 and anti-IL-18 are
bispecific
antibodies and are administered to a patient for treatment of disease.
Examples of diseases
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include immune diseases and autoimmune diseases, and include inflammatory
bowel disease
(IBD), age-related macular degeneration (AMD), and type 2 diabetes (T2D).
[0048] In some embodiments, the anti-IL-1[3 and anti-IL-18 antibodies of the
present
invention, block or neutralize the activity of, and/or bind to, IL-113 and/or
IL-18.
[0049] All references, including patents, applications, and scientific
literature, cited herein
are hereby incorporated by reference in their entirety.
GENERAL TECHNIQUES
[0050] The techniques and procedures described or referenced herein are
generally well
understood and commonly employed using conventional methodology by those
skilled in the
art, such as, for example, the widely utilized methodologies described in
Sambrook et al.,
Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M.
Ausubel, et al.
eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2:
A Practical
Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow
and Lane,
eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I.
Freshney, ed.
(1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in
Molecular Biology,
Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998)
Academic Press;
Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and
Tissue Culture (J. P.
Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:
Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J.
Wiley and Sons;
Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.);
Gene
Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Cabs, eds.,
1987); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in
Immunology (J.
E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley
and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch,
1997);
Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);
Monoclonal
Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford
University Press,
2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold
Spring Harbor
Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds.,
Harwood
Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology
(V. T. DeVita
et al., eds., J.B. Lippincott Company, 1993).
DEFINITIONS
[0051] For purposes of interpreting this specification, the following
definitions will apply
and whenever appropriate, terms used in the singular will also include the
plural and vice
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versa. In the event that any definition set forth below conflicts with any
document incorporated
herein by reference, the definition set forth below shall control.
[0052] With reference to the molecules referred to herein (e.g., antibodies,
and the
molecules that the antibodies bind to), "active" or "activity" refer to
biological, immunological
and/or functional activities of such molecules. For example, in some
embodiments, the anti-
IL-1[3 and anti-IL-18 antibodies of the present invention are bispecific
antibodies that bind to
IL-113 and IL-18 and thus have a binding activity. In a further embodiment,
anti-IL-113 and anti-
IL-18 antibodies of the present invention have neutralizing or blocking
activity, i.e., such
antibodies can neutralize or block the activity of IL-113 and/or IL-18.
[0053] "Affibodies" or "Affibody" refers to the use of a protein liked by
peptide bond to an
Fc region, wherein the protein is used as a scaffold to provide a binding
surface for a target
molecule. The binding surface may be altered through mutagenisis to generate a
library of
proteins that can bind other target molecules or other epitopes on the same
target molecule.
The starting protein is often a naturally occurring protein such as
staphylococcal protein A or
IgG-binding B domain, or the Z protein derived therefrom (see Nilsson et al
(1987), Prot Eng
1, 107-133, and U.S. Pat. No. 5,143,844) or a fragment or derivative thereof.
For example,
affibodies can be created from Z proteins variants having altered binding
affinity to target
molecule(s), wherein a segment of the Z protein has been mutated by random
mutagenesis
to create a library of variants capable of binding a target molecule. Examples
of affibodies
include U.S. Pat. No. 6,534,628, Nord K et al, Prot Eng 8:601-608 (1995) and
Nord K et al,
Nat Biotech 15:772-777 (1997). Biotechnol Appl Biochem. 2008 Jun; 50(Pt 2):97-
112.
[0054] The term "antibody" herein is used in the broadest sense and refers to
any
immunoglobulin (Ig) molecule whether naturally occurring or engineered, and
any fragment,
mutant, variant or derivation thereof which so long as it exhibits the desired
biological activity
(e.g., epitope binding activity). Examples of antibodies include, but are not
limited to,
monoclonal antibodies, polyclonal antibodies, multispecific antibodies,
antibody fragments,
single domain antibodies, octopus antibodies and DVD antibodies. In one
embodiment, an
antibody of the present invention comprises at least one variable domain. In
another
embodiment, an antibody of the present invention is a bispecific antibody.
[0055] Generally, immunoglobulins are assigned to different classes, depending
on the
amino acid sequences of the heavy chain constant domains. Five major classes
of
immunoglobulins have been described: IgA, IgD, IgE, IgG and IgM. These may be
further
divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgA-1, IgA-2, and the
like. The heavy
chain constant domains corresponding to each immunoglobulin class are termed
a, 6, E, y
and p for IgA, D, E, G, and M, respectively. The subunit structures and three-
dimensional
configurations of the different classes of immunoglobulins are well known and
described
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generally, for example, in Abbas et al., 2000, Cellular and Mol. Immunology,
4th ed. An
antibody may be part of a larger fusion molecule, formed by covalent or non-
covalent
association of the antibody with one or more other protein or peptide.
[0056] In one embodiment, antibodies of the present invention have reduced
(fewer)
disulfide linkages. In one embodiment, antibodies of the invention comprise a
hinge region in
which at least one cysteine residue is rendered incapable of forming a
disulfide linkage,
wherein the disulfide linkage is preferably intermolecular, preferably between
two heavy
chains. A hinge cysteine can be rendered incapable of forming a disulfide
linkage by any of a
variety of suitable methods known in the art, some of which are described
herein, including
but not limited to deletion of the cysteine residue or substitution of the
cysteine with another
amino acid.
[0057] The phrase "an anti- IL-113 antibody and/or anti-IL-18
antibody/antibodies" refers,
depending on the context, to (1) an anti- IL-1[3 antibody, or (2) an anti-IL-
18 antibody, or (3) a
combination of an anti- IL-113 antibody and an anti-IL-18 antibody (i.e., two
antibodies), or (4)
an antibody that binds to both IL-113 and IL-18.
[0058] An "affinity matured" antibody is one having one or more alteration in
one or more
CDRs thereof which result in an improvement in the affinity of the antibody
for antigen,
compared to a parent antibody which does not possess those alteration(s).
Preferred affinity
matured antibodies will have nanomolar or even picomolar affinities for the
target antigen.
Affinity matured antibodies are produced by known procedures. See, for
example, Marks et
al., 1992, Biotechnology 10:779-783 that describes affinity maturation by
variable heavy chain
(VH) and variable light chain (VL) domain shuffling. Random mutagenesis of CDR
and/or
framework residues is described in: Barbas, et al. 1994, Proc. Nat. Acad. Sci,
USA 91:3809-
3813; Shier et al., 1995, Gene 169:147-155; YeIton et al., 1995, J. Immunol.
155:1994-2004;
Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al, 1992, J.
Mol. Biol.
226:889-896, for example.
[0059] An "agonist antibody" or "agonistic antibody" is an antibody that
binds and
activates an antigen, such as a receptor. Generally, receptor activation
capability of the
agonist antibody will be at least qualitatively similar (and may be
essentially quantitatively
similar) to that of a native agonist ligand of the receptor.
[0060] "Antibody fragments" refers to an antibody comprising a portion of an
intact
antibody, preferably the antigen binding or a variable region of the intact
antibody. Examples
of antibody fragments include Fab, Fab', F(ab)2, and Fv fragments; diabodies
(Db); tandem
diabodies (taDb), linear antibodies (see U.S. Patent No. 5,641,870, Example 2;
Zapata et al.,
Protein Eng. 8(10):1057-1062 (1995)); one-armed antibodies, minibodies, single-
chain
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antibody molecules; and multispecific antibodies formed from antibody
fragments (e.g.,
including but not limited to, Db-Fc, taDb-Fc, taDb-CH3 and (scFV)4-Fc).
[0061] In some embodiments, an antibody fragment comprises only a portion of
an intact
antibody, where the portion retains at least one, and may retain most or all,
of the functions
normally associated with that portion when present in an intact antibody. In
another embody,
an antibody fragment of the invention comprises a sufficient portion of the
constant region to
permit dimerization (or multimerization) of heavy chains that have reduced
disulfide linkage
capability, for example where at least one of the hinge cysteines normally
involved in inter-
heavy chain disulfide linkage is altered as described herein. In one
embodiment, an antibody
fragment comprises an antigen binding site or variable domains of the intact
antibody and
thus retains the ability to bind antigen. In another embodiment, an antibody
fragment, for
example one that comprises the Fc region, retains at least one of the
biological functions
normally associated with the Fc region when present in an intact antibody,
such as FcRn
binding, antibody half life modulation, ADCC function, and/or complement
binding (for
example, where the antibody has a glycosylation profile necessary for ADCC
function or
complement binding). Examples of antibody fragments include, but are not
limited to, linear
antibodies; single-chain antibody molecules; and multispecific antibodies
formed from
antibody fragments.
[0062] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a
cell-
mediated reaction in which nonspecific cytotoxic cells that express FcRs (such
as Natural
Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a
target cell
and subsequently cause lysis of the target cell. NK cells, the primary cells
for mediating
ADCC, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and
FcyRIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page 464 of
Ravetch et al.,
1991, Annu. Rev. Immunol 9:457-92. To assess ADCC activity of a molecule of
interest, an in
vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or
5,821,337 may be
performed. Useful effector cells for such assays include peripheral blood
mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the
molecule of interest may be assessed in vivo, for example, in a animal model
such as that
disclosed in Clynes et al., 1998, PNAS (USA) 95:652-656.
[0063] The terms "anti- IL-113 antibody" and "an antibody that binds to IL-1
13" refer to an
antibody that is capable of binding IL-113 with sufficient affinity such that
the antibody is useful
as a diagnostic and/or therapeutic agent in targeting IL-1 13. In one
embodiment, the extent of
binding of an anti- IL-16 antibody to an unrelated, non- IL-16 protein is less
than about 10% of
the binding of the antibody to IL-113 as measured, e.g., by a radioimmunoassay
(RIA). In
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certain embodiments, an anti- IL-113 antibody binds to an epitope of IL-1[3
that is conserved
among IL-113 from different species.
[0064] The terms "anti-IL-18 antibody" and "an antibody that binds to IL-18"
refer to an
antibody that is capable of binding IL-18 with sufficient affinity such that
the antibody is useful
as a diagnostic and/or therapeutic agent in targeting IL-18. In one
embodiment, the extent of
binding of an anti-IL-18 antibody to an unrelated, non-IL-18 protein is less
than about 10% of
the binding of the antibody to IL-18 as measured, e.g., by a radioimmunoassay
(RIA). In
certain embodiments, an anti-IL-18 antibody binds to an epitope of IL-18 that
is conserved
among IL-18 from different species.
[0065] An "autoimmune disease" as used herein is a non-malignant disease or
disorder
arising from and directed against an individual's own tissues. The autoimmune
diseases
described herein specifically exclude malignant or cancerous diseases or
conditions,
particularly excluding B cell lymphoma, acute lymphoblastic leukemia (ALL),
chronic
lymphocytic leukemia (CLL), Hairy cell leukemia, and chronic myeloblastic
leukemia.
Examples of autoimmune diseases or disorders include, but are not limited to,
age-related
macular degeneration (AMD), inflammatory responses such as inflammatory skin
diseases
including psoriasis and dermatitis (for example, atopic dermatitis); systemic
scleroderma and
sclerosis; responses associated with inflammatory bowel disease (such as
Crohn's disease
and ulcerative colitis); respiratory distress syndrome (including adult
respiratory distress
syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis;
glomerulonephritis;
allergic conditions such as eczema and asthma and other conditions involving
infiltration of T
cells and chronic inflammatory responses; atherosclerosis; leukocyte adhesion
deficiency;
rheumatoid arthritis; systemic lupus erythematosus (SLE); lupus nephritis
(LN); diabetes
mellitus (e.g. Type I diabetes mellitus or insulin dependent diabetes
mellitis); multiple
sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic
encephalomyelitis; Sjorgen's
syndrome; juvenile onset diabetes; and immune responses associated with acute
and
delayed hypersensitivity mediated by cytokines and T-lymphocytes typically
found in
tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis;
pernicious anemia
(Addison's disease); diseases involving leukocyte diapedesis; central nervous
system (CNS)
inflammatory disorder; multiple organ injury syndrome; hemolytic anemia
(including, but not
limited to cryoglobinemia or Coombs positive anemia); myasthenia gravis;
antigen-antibody
complex mediated diseases; anti-glomerular basement membrane disease;
antiphospholipid
syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic
syndrome;
pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter's
disease; stiff-
man syndrome; Behcet disease; giant cell arteritis; immune complex nephritis;
IgA
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nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or
autoimmune
thrombocytopenia etc.
[0066] AMD is a leading cause of severe, irreversible vision loss among the
elderly (see
e.g., Bressler (2004) JAMA 291:1900-01). It is characterized by a broad
spectrum of clinical
and pathologic findings, including pale yellow spots known as drusen,
disruption of the retinal
pigment epithelium (RPE), choroidal neovascularization (CNV), and disciform
macular
degeneration. The manifestations of the disease is classified into two forms:
non-exudative
(dry) and exudative (wet or neovascular). Recently, several therapies for
treatment of wet
AMD have been approved ¨ photodynamic therapy using verteporfin (Visudyne0); a
VEGF-
binding aptamer, pegaptantib (Macugen0); and an anti-VEGF antibody fragment,
ranibizumab (Lucentis0).
[0067] An "autoinflammatory disease" as used herein refers to a group of rare
hereditary
immune-mediated disorders that share similar features, particularly fever.
Autoinflammatory
diseases are characterized by recurrent unprovoked inflammation in the absence
of high titers
of autoantibodies, infection, or antigen-specific T lymphocytes. Exemplary
autoinflammatory
diseases include, but is not limited to, Familial Mediterranean Fever (FMF);
tumour necrosis
factor (TNF) receptor-associated periodic fever syndrome (TRAPS);
hyperimmunoglobulinemia D and periodic fever syndrome (HIDS); systemic onset
juvenile
idiopathic arthritis (Still's disease); cryopyrin-associated periodic syndrome
(CAPS); familial
cold autoinflammatory syndrome; Muckle-Wells syndrome; deficiency of the
interleukin-1
receptor antagonist (DIRA); and neonatal onset multi-system inflammatory
disease
(NOMID)/chronic infantile neurological cutaneous and articular (CINCA)
syndrome.
[0068] Autoimmune and autoinflammatory diseases share common characteristics
in that
both groups of disorders result from the immune system attacking the body's
own tissues,
and also result in increased inflammation. The distinguishing feature between
the two is the
lack of autoantibodies (at high titers) in autoinflammatory diseases.
[0069] A "biologically active" or "functional" immunoglobulin is one capable
of exerting
one or more of its natural activities in structural, regulatory, biochemical
or biophysical events.
For example, a biologically active antibody may have the ability to
specifically bind an antigen
and the binding may elicit or alter a cellular or molecular event such as
signaling transduction
or enzymatic activity. A biologically active antibody may also block ligand
activation of a
receptor or act as an agonist antibody. The capability of an antibody to exert
one or more of
its natural activities depends on several factors, including proper folding
and assembly of the
polypeptide chains.
[0070] "Binding affinity" generally refers to the strength of the sum total of
noncovalent
interactions between a single binding site of a molecule (e.g., an antibody)
and its binding
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partner (e.g., an antigen). The affinity of a molecule X for its partner Y can
generally be
represented by the dissociation constant (Kd). Affinity can be measured by
common methods
known in the art, including those described herein. Low-affinity antibodies
bind antigen
weakly and tend to dissociate readily, whereas high-affinity antibodies bind
antigen more
tightly and remain bound longer.
[0071] "Biological molecule" refers to a nucleic acid, a protein, a
carbohydrate, a lipid,
and combinations thereof. In one embodiment, the biologic molecule exists in
nature.
[0072] A "blocking" antibody or an "antagonist" antibody is one that inhibits
or reduces
biological activity of the antigen it binds. Such blocking can occur by any
means, for example,
by interfering with: ligand binding to the receptor, receptor complex
formation, tyrosine kinase
activity of a tyrosine kinase receptor in a receptor complex and/or
phosphorylation of tyrosine
kinase residue(s) in or by the receptor. Preferred blocking antibodies or
antagonist antibodies
substantially or completely inhibit the biological activity of the antigen.
[0073] The terms "cancer" and "cancerous" refer to or describe the
physiological
condition in mammals that is typically characterized by unregulated cell
growth. Examples of
cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,
and
leukemia. More particular examples of such cancers include squamous cell
cancer, small-cell
lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous
carcinoma of
the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal
cancer, pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder
cancer,
hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or
uterine carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval
cancer, thyroid
cancer, hepatic carcinoma, and various types of head and neck cancer.
[0074] The term "chimeric" antibodies refer to antibodies in which a portion
of the heavy
and/or light chain is identical with or homologous to corresponding sequences
in antibodies
derived from a particular species or belonging to a particular antibody class
or subclass, while
the remainder of the chain(s) is identical with or homologous to corresponding
sequences in
antibodies derived from another species or belonging to another antibody class
or subclass,
as well as fragments of such antibodies, so long as they exhibit the desired
biological activity
(See, for example, U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc.
Natl. Acad. Sci.
USA 81:6851-6855).
[0075] As used herein, the expressions "cell," "cell line," and "cell culture"
are used
interchangeably and all such designations include progeny. Thus, the words
"transformants"
and "transformed cells" include the primary subject cell and cultures derived
therefrom without
regard for the number of transfers. It is also understood that all progeny may
not be precisely
identical in DNA content, due to deliberate or inadvertent mutations. Mutant
progeny that
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have the same function or biological activity as screened for in the
originally transformed cell
are included. Where distinct designations are intended, it will be clear from
the context.
[0076] The expression "control sequences" refers to DNA sequences necessary
for the
expression of an operably linked coding sequence in a particular host
organism. The control
sequences that are suitable for prokaryotes, for example, include a promoter,
optionally an
operator sequence, and a ribosome binding site. Eukaryotic cells are known to
utilize
promoters, polyadenylation signals, and enhancers.
[0077] "Diabetes" as used herein is a chronic disorder affecting carbohydrate,
fat and
protein metabolism in animals. Diabetes is the leading cause of blindness,
renal failure, and
lower limb amputations in adults and is a major risk factor for cardiovascular
disease and
stroke. Type I diabetes mellitus (or insulin-dependent diabetes mellitus
("IDDM") or juvenile-
onset diabetes) comprises approximately 10% of all diabetes cases. The disease
is
characterized by a progressive loss of insulin secretory function by beta
cells of the pancreas.
This characteristic is also shared by non-idiopathic, or "secondary", diabetes
having its origins
in pancreatic disease. Type I diabetes mellitus is associated with the
following clinical signs or
symptoms, e.g., persistently elevated plasma glucose concentration or
hyperglycemia;
polyuria; polydipsia and/or hyperphagia; chronic microvascular complications
such as
retinopathy, nephropathy and neuropathy; and macrovascular complications such
as
hyperlipidemia and hypertension which can lead to blindness, end-stage renal
disease, limb
amputation and myocardial infarction.
[0078] Type II diabetes mellitus (non-insulin-dependent diabetes mellitus or
NIDDM) is a
metabolic disorder involving the dysregulation of glucose metabolism and
impaired insulin
sensitivity. Type II diabetes mellitus usually develops in adulthood and is
associated with the
body's inability to utilize or make sufficient insulin. In addition to the
insulin resistance
observed in the target tissues, patients suffering from type II diabetes
mellitus have a relative
insulin deficiency--that is, patients have lower than predicted insulin levels
for a given plasma
glucose concentration. Type II diabetes mellitus is characterized by the
following clinical signs
or symptoms, e.g., persistently elevated plasma glucose concentration or
hyperglycemia;
polyuria; polydipsia and/or hyperphagia; chronic microvascular complications
such as
retinopathy, nephropathy and neuropathy; and macrovascular complications such
as
hyperlipidemia and hypertension which can lead to blindness, end-stage renal
disease, limb
amputation and myocardial infarction. Syndrome X, also termed Insulin
Resistance
Syndrome (IRS), Metabolic Syndrome, or Metabolic Syndrome X, is recognized in
some 2%
of diagnostic coronary catheterizations. Often disabling, it presents symptoms
or risk factors
for the development of Type II diabetes mellitus and cardiovascular disease,
including, e.g.,
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impaired glucose tolerance (IGT), impaired fasting glucose (IFG),
hyperinsulinemia, insulin
resistance, dyslipidemia (e.g., high triglycerides, low HDL), hypertension and
obesity.
[0079] A "disorder" is any condition that would benefit from treatment with a
therapeutic
antibody or antibodies. This includes chronic and acute disorders or diseases
including those
pathological conditions which predispose the mammal to the disorder in
question. In some
embodiments, the disorder is a cancer, an inflammatory, an immune, an
autoinflammatory or
an autoimmune disease.
[0080] An "extracellular domain" is defined herein as that region of a
transmembrane
polypeptide, such as an FcR, that is external to a cell.
[0081] The terms "Fe receptor" or "FeR" are used to describe a receptor that
binds to the
Fc region of an antibody.
[0082] The term "Fe region" herein is used to define a C-terminal region of an
immunoglobulin heavy chain, including native sequence Fc regions and variant
Fc regions. In
one embodiment, the Fc region comprises a CH2 domain and/or a CH3 domain.
Although
the boundaries of the Fc region of an immunoglobulin heavy chain might vary,
the human IgG
heavy chain Fc region is usually defined to stretch from an amino acid residue
at position
Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal
lysine (residue 447
according to the EU numbering system) of the Fc region may be removed, for
example,
during production or purification of the antibody, or by recombinantly
engineering the nucleic
acid encoding a heavy chain of the antibody. Accordingly, a composition of
intact antibodies
may comprise antibody populations with all K447 residues removed, antibody
populations with
no K447 residues removed, and antibody populations having a mixture of
antibodies with and
without the K447 residue. "Fe complex" as used herein refers to two CH2
domains and/or two
CH3 domains, wherein the CH2 domains and/or the CH3 domains are bound together
through interactions that are not peptide bonds.
[0083] "Framework regions" (FR) are those variable domain residues other than
the CDR
residues. Each IgG variable domain typically has four FRs identified as FR1,
FR2, FR3, and
FR4. If the CDRs are defined according to Kabat, the light chain FR residues
are positioned
at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107
(LCFR4) and
the heavy chain FR residues are positioned about at residues 1-30 (HCFR1), 36-
49 (HCFR2),
66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the CDRs
comprise
amino acid residues from hypervariable loops, the light chain FR residues are
positioned
about at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107
(LCFR4) in the
light chain and the heavy chain FR residues are positioned about at residues 1-
25 (HCFRI),
33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues.
In some
instances, when the CDR comprises amino acids from both a CDR as defined by
Kabat and
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those of a hypervariable loop, the FR residues will be adjusted accordingly.
For example,
when CDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are at
positions
1-25 and the FR2 residues are at positions 36-49.
[0084] A "functional Fc region" possesses an "effector function" of a native
sequence Fc
region. Exemplary "effector functions" include C1q binding; complement
dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor;
BCR), and the
like. Such effector functions generally require the Fc region to be combined
with a binding
domain (e.g. an antibody variable domain) and can be assessed using various
assays as, for
example, those disclosed herein. A "native sequence Fc region" comprises an
amino acid
sequence identical to the amino acid sequence of a Fc region found in nature.
Native
sequence human Fc regions include a native sequence human IgG1 Fc region (non-
A and A
allotypes); native sequence human IgG2 Fc region; native sequence human IgG3
Fc region;
and native sequence human IgG4 Fc region as well as naturally occurring
variants thereof. A
"variant Fc region" comprises an amino acid sequence that differs from a
native sequence Fc
region by virtue of at least one "amino acid modification" as herein defined.
The variant Fc
region can have at least one amino acid substitution compared to a native
sequence Fc
region or to the Fc region of a parent antibody, and may have, for example,
from about one to
about ten amino acid substitutions, or from about one to about five amino acid
substitutions in
a native sequence Fc region or in the Fc region of the parent antibody. The
variant Fc region
can possess at least about 80% identity with a native sequence Fc region
and/or with an Fc
region of a parent antibody, and may have at least about 90% identity
therewith, or have at
least about 95% identity therewith.
[0085] The terms "full length antibody," "intact antibody" and "whole
antibody" are used
herein interchangeably, to refer to an antibody in its substantially intact
form, and not antibody
fragments as defined below. The terms particularly refer to an antibody with
heavy chains
and Fc regions. An antibody variant of the invention can be for example a full
length
antibody. Also, a full length antibody can be for example human, humanized,
chimeric, and/or
affinity matured.
[0086] A "hinge region," and variations thereof, as used herein, includes the
meaning
known in the art, which is illustrated in, for example, Janeway et al., 1999,
Immuno Biology:
The Immune System in Health and Disease, Elsevier Science Ltd., NY. 4th ed.;
Bloom et al.,
1997, Protein Science, 6:407-415; Humphreys et al., 1997, J. Immunol. Methods,
209:193-
202.
[0087] "Homology" is defined as the percentage of residues in the amino acid
sequence
variant that are identical after aligning the sequences and introducing gaps,
if necessary, to
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achieve the maximum percent homology. Methods and computer programs for the
alignment
are well known in the art. One such computer program is "Align 2," authored by
Genentech,
Inc., and filed with user documentation in the United States Copyright Office,
Washington,
D.C. 20559, on Dec. 10, 1991.
[0088] The term "host cell" (or "recombinant host cell"), as used herein,
refers to a cell
that has been genetically altered, or is capable of being genetically altered,
by introduction of
an exogenous polynucleotide, such as a recombinant plasmid or vector. It
should be
understood that such terms are intended to refer not only to the particular
subject cell but to
the progeny of such a cell. Because certain modifications may occur in
succeeding
generations due to either mutation or environmental influences, such progeny
may not, in
fact, be identical to the parent cell, but are still included within the scope
of the term "host cell"
as used herein.
[0089] A "human consensus framework" is a framework that represents the most
commonly occurring amino acid residues in a selection of human immunoglobulin
VI_ or VH
framework sequences. Generally, the selection of human immunoglobulin VI_ or
VH
sequences is from a subgroup of variable domain sequences. Generally, the
subgroup of
sequences is a subgroup as in Kabat. In one embodiment, for the VL, the
subgroup is
subgroup kappa I as in Kabat. In one embodiment, for the VH, the subgroup is
subgroup III
as in Kabat.
[0090] The Kabat numbering system is generally used when referring to a
residue in the
variable domain (approximately residues 1-107 of the light chain and residues
1-113 of the
heavy chain) (e.g, Kabat etal., Sequences of Immunological Interest. 5th Ed.
Public Health
Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU
numbering system" or
"EU index" is generally used when referring to a residue in an immunoglobulin
heavy chain
constant region (e.g., the EU index reported in Kabat etal., supra). The "EU
index as in
Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless
stated
otherwise herein, references to residue numbers in the variable domain of
antibodies means
residue numbering by the Kabat numbering system. Unless stated otherwise
herein,
references to residue numbers in the constant domain of antibodies means
residue
numbering by the EU numbering system (e.g., see United States Provisional
Application No.
60/640,323, Figures for EU numbering).
[0091] A naturally occurring basic 4-chain antibody unit is a heterotetrameric
glycoprotein
composed of two identical light (L) chains and two identical heavy (H) chains
(an IgM antibody
consists of 5 of the basic heterotetramer units along with an additional
polypeptide called J
chain, and therefore contains 10 antigen binding sites, while secreted IgA
antibodies can
polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain
units along
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with J chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each
L chain is linked to an H chain by one covalent disulfide bond, while the two
H chains are
linked to each other by one or more disulfide bonds depending on the H chain
isotype. Each
H and L chain also has regularly spaced intrachain disulfide bridges. Each H
chain has, at
the N-terminus, a variable domain (VH) followed by three constant domains (CH)
for each of
the a and y chains and four CH domains for p and E isotypes. Each L chain has,
at the N-
terminus, a variable domain (VL) followed by a constant domain (CL) at its
other end. The VL
is aligned with the VH and the CL is aligned with the first constant domain of
the heavy chain
(CH1). Particular amino acid residues are believed to form an interface
between the light
chain and heavy chain variable domains. The pairing of a VH and VL together
forms a single
antigen-binding site. For the structure and properties of the different
classes of antibodies,
see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba
I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and
Chapter 6.
[0092] The L chain from any vertebrate species can be assigned to one of two
clearly
distinct types, called kappa and lambda, based on the amino acid sequences of
their constant
domains. Depending on the amino acid sequence of the constant domain of their
heavy
chains (CH), immunoglobulins can be assigned to different classes or isotypes.
There are
five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy
chains designated
a, 6, y, E, and p, respectively. The y and a classes are further divided into
subclasses on the
basis of relatively minor differences in CH sequence and function, e.g.,
humans express the
following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
[0093] "Human effector cells" are leukocytes that express one or more FcRs and
perform
effector functions. In some embodiments the cells express at least FcyRIII and
perform
ADCC effector function. Examples of human leukocytes that mediate ADCC include
peripheral blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T
cells, and neutrophils. The effector cells may be isolated from a native
source, for example,
from blood or PBMCs (Peripheral blood mononuclear cells) as described herein.
[0094] "Humanized" forms of non-human (for example, murine) antibodies are
chimeric
antibodies that contain 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
nonhuman primate having the desired specificity, affinity, and capacity. In
some instances, Fv
framework region (FR) residues of the human immunoglobulin are replaced by
corresponding
non-human residues. Furthermore, humanized antibodies may comprise residues
that are not
found in the recipient antibody or in the donor antibody. These modifications
are made to
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further refine antibody performance. In general, the humanized an body 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 regions are those of a
human
immunoglobulin sequence. The humanized antibody optionally also will comprise
at least a
portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
For further details, see 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).
[0095] A "human antibody" is an antibody that possesses an amino acid sequence
corresponding to that of an antibody produced by a human and/or has been made
using any
of the techniques for making human antibodies disclosed herein. This
definition specifically
excludes a humanized antibody that comprises non-human antigen-binding
residues.
[0096] As used herein, the term "hyperglycemic disorders" refers to all forms
of diabetes
and disorders resulting from insulin resistance, such as Type I and Type ll
diabetes, as well
as severe insulin resistance, hyperinsulinemia, and hyperlipidemia, e.g.,
obese subjects, and
insulin-resistant diabetes, such as Mendenhall's Syndrome, Werner Syndrome,
leprechaunism, lipoatrophic diabetes, and other lipoatrophies. A particular
hyperglycemic
disorder disclosed herein is diabetes, especially Type 1 and Type ll diabetes.
"Diabetes" itself
refers to a progressive disease of carbohydrate metabolism involving
inadequate production
or utilization of insulin and is characterized by hyperglycemia and
glycosuria.
[0097] The term "hypervariable region," "HVR," or "HV," when used herein
refers to the
regions of an antibody variable domain which are hypervariable in sequence
and/or form
structurally defined loops. Generally, antibodies comprise six HVRs; three in
the VH (H1, H2,
H3), and three in the VI_ (L1, L2, L3). In native antibodies, H3 and L3
display the most
diversity of the six HVRs, and H3 in particular is believed to play a unique
role in conferring
fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45
(2000); Johnson and
Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa,
NJ, 2003).
However, there are a number of examples of naturally occurring and engineered,
functional
antibodies having only a heavy chain and lacking a light chain. See, e.g.,
Hamers-Casterman
et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-
736 (1996).
[0098] A number of HVR delineations are in use and are encompassed herein. The
Kabat Complementarity Determining Regions (CDRs) are based on sequence
variability and
are the most commonly used (Kabat et al., Sequences of Proteins of
Immunological Interest,
5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
(1991)). Chothia
refers instead to the location of the structural loops (Chothia and Lesk J.
Mol. Biol. 196:901-
917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and
Chothia
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structural loops, and are used by Oxford Molecular's AbM antibody modeling
software. The
"contact" HVRs are based on an analysis of the available complex crystal
structures. The
residues from each of these HVRs are noted below.
Loop Kabat AbM Chothia Contact
L1 L24-L34 L24-L34 L26-L32 L30-L36
L2 L50-L56 L50-L56 L50-L52 L46-L55
L3 L89-L97 L89-L97 L91-L96 L89-L96
H1 (Kabat
H31-H35B H26-H35B H26-H32 H30-H35B
Numbering)
H1 (Chothia
H31-H35 H26-H35 H26-H32 H30-H35
Numbering)
H2 H50-H65 H50-H58 H53-H55 H47-H58
H3 H95-H102 H95-H102 H96-H101 H93-H101
[0099] HVRs generally comprise amino acid residues from the hypervariable
loops
and/or from the "complementarity determining regions" (CDRs), the latter being
of highest
sequence variability and/or involved in antigen recognition. Exemplary
hypervariable loops
occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1),
53-55 (H2), and
96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary
CDRs (CDR-
L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues
24-34
of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3.
(Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National
Institutes of Health, Bethesda, MD (1991).) With the exception of CDR1 in VH,
CDRs
generally comprise the amino acid residues that form the hypervariable loops.
CDRs also
comprise "specificity determining residues," or "SDRs," which are residues
that contact
antigen. SDRs are contained within regions of the CDRs called abbreviated-
CDRs, or a-
CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-
CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-
35B of H1,
50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci.
13:1619-1633
(2008).) IgG HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or
50-56 (L2) and 89-97 or 89-96 (L3) in the VI_ and 26-35 (H1), 50-65 or 49-65
(H2) and 93-102,
94-102, or 95-102 (H3) in the VH. Unless otherwise indicated, HVR residues and
other
residues in the variable domain (e.g., FR residues) are numbered herein
according to Kabat
et al., supra.
[0100] Antibodies having a VH/VL unit that can bind two or more epitopes can
be made
(Bostrom et al. (2009) Science 323:1610-1614; WO 2008/027236 (incorporated by
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reference). Such multispecific antibodies are referred to herein as "two-in
one" antibodies or
"dual acting Fab" or "DAF" to indicate that a single arm of an antibody (aka
the VH/VL unit) can
bind to at least two epitopes on the same target molecule or two epitopes on
different target
molecules. In one aspect, these DAF antibodies can be made by mutating the VI_
domain of a
VH/VL unit of an antibody that binds a first epitope and selecting the mutant
VH/VL unit that can
bind the first epitope and a second epitope. For example, in one embodiment,
one or more
solvent accessible amino acid residue(s) of the light chain CDRs are be
randomly or
selectively substituted with one or more other amino acid residues(s) prior to
screening the
mutated VH/VL unit for binding to a second epitope.
[0101] An "inflammasome-mediated disease" refers to any disease where IL-113
and/or
IL-18 are elevated relative to normal, uninflammed tissue. Generally, in an
inflammasome-
mediated disease, caspase-1 processing and/or activation is involved/elevated
relative to
uninduced control cells. Caspase-1 activity can be measured using commercially
available
assay kits, e.g., Caspase 1 Fluorometric Assay Kit ((Cat. No. ab394120; AbCam,
Cambridge,
MA), Caspase-1 Colorimetric Assay (Cat. No. BF14100; R&D Systems), etc.
[0102] In general, a disease or condition can be considered an IL-1[3 related
disease or
condition if it is associated with elevated levels of IL-1[3 in bodily fluids
or tissue or if cells or
tissues taken from the body produce elevated levels of IL-113 in culture.
Similarly, a disease
or condition can be considered an IL-18 related disease or condition if it is
associated with
elevated levels of IL-18 in bodily fluids or tissue or if cells or tissues
taken from the body
produce elevated levels of IL-18 in culture. Thus, an IL-18/IL-18 related
disease or condition
is associated with elevated levels of IL-113 and IL-18 in bodily fluids or
tissue or if cells or
tissues taken from the body produce elevated levels of both cytokines in
culture.
[0103] Immune and inflammatory diseases include: chronic obstructive pulmonary
disease (COPD), rheumatoid arthritis, osteoarthritis, juvenile chronic
arthritis,
spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic
inflammatory myopathies
(dermatomyositis), systemic vasculitis, sarcoidosis, autoimmune hemolytic
anemia (immune
pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune
thrombocytopenia
(idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia),
thyroiditis
(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis,
atrophic thyroiditis)
autoimmune inflammatory diseases (e.g., allergic encephalomyelitis, multiple
sclerosis,
insulin-dependent diabetes mellitus, autoimmune uveoretinitis, thyrotoxicosis,
autoimmune
thyroid disease, pernicious-anemia, autograft rejection, diabetes mellitus,
and immune-
mediated renal disease (glomerulonephritis, tubulointerstitial nephritis)),
demyelinating
diseases of the central and peripheral nervous systems such as multiple
sclerosis, idiopathic
demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic
inflammatory
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demyelinating polyneuropathy; hepatobiliary diseases such as infectious
hepatitis (hepatitis A,
B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active
hepatitis, primary
biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, gluten-
sensitive
enteropathy, and Whipple's disease; autoimmune or immune-mediated skin
diseases
including bullous skin diseases, erythema multiforme and contact dermatitis,
psoriasis;
allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, vernal
conjunctivitis,
eczema, food hypersensitivity and urticaria; immunologic diseases of the lung
such as
eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity
pneumonitis;
transplantation associated disease including graft rejection and graft-versus-
host-disease.
[0104] Immune related and inflammatory diseases are the manifestations or
consequences of fairly complex, often multiple interconnected biological
pathways which in
normal physiology are critical to respond to insult or injury, initiate repair
from insult or injury,
and mount innate and acquired defense against foreign organisms. Disease or
pathology
occurs when these normal physiological pathways cause additional insult or
injury either as
directly related to the intensity of the response, as a consequence of
abnormal regulation or
excessive stimulation, as a reaction to self, or a combination of these.
[0105] Examples of IL-113 related diseases are acute pancreatitis; ALS;
cachexia/anorexia, including AIDS-induced cachexia; asthma and other pulmonary
diseases;
autoimmune vasculitis; CIAS1 Associated Periodic Syndromes (CAPS); Neonatal
Onset
Multisystem Inflammatory Disorder (NOMID/CINCA), systemic onset juvenile
idiopathic
arthritis, Stills disease, Muckle-Wells syndrome, chronic fatigue syndrome;
Clostridium
associated illnesses, including Clostridium-associated diarrhea; coronary
conditions and
indications, including congestive heart failure, coronary restenosis,
myocardial infarction,
myocardial dysfunction (e.g., related to sepsis), and coronary artery bypass
graft; cancers,
such as multiple myeloma and myelogenous (e.g., AML and CML) and other
leukemias, as
well as tumor metastasis; diabetes (e.g., insulin diabetes); endometriosis;
familial Cold
Autoinflammatory Syndrome (FCAS); familial mediterranean fever (FMF); fever;
fibromyalgia;
glomerulonephritis; graft versus host disease/transplant rejection;
hemohorragic shock;
hyperalgesia; inflammatory bowel disease; inflammatory conditions of a joint,
including
psoriatic arthritis (as well as osteoarthritis and rheumatoid arthritis);
inflammatory eye
disease, as may be associated with, for example, corneal transplant; ischemia,
including
cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy,
hemorrhage or stroke,
each of which may lead to neurodegeneration); Kawasaki's disease; learning
impairment;
lung diseases (e.g., ARDS); myopathies (e.g., muscle protein metabolism,
especially in
sepsis); neurotoxicity (e.g., as induced by HIV); osteoporosis; pain,
including cancer-related
pain; Parkinson's disease; periodontal disease; pre-term labor; psoriasis;
reperfusion injury;
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side effects from radiation therapy; sleep disturbance; temporal mandibular
joint disease;
tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS);
uveitis; or an
inflammatory condition resulting from strain, sprain, cartilage damage,
trauma, orthopedic
surgery, infection or other disease processes.
[0106] Interleukin 18 plays a critical role in the pathology associated with a
variety of
diseases involving immune and inflammatory elements. These diseases include,
but are not
limited to, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis,
Lyme arthritis, psoriatic
arthritis, reactive arthritis, spondyloarthropathy, lupus (e.g., Systemic
Lupus Erythematosus,
and Lupus Nephritis), Crohn's disease, ulcerative colitis, inflammatory bowel
disease, insulin
dependent diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, psoriasis type 1,
psoriasis type 2, dermatitis scleroderma, graft versus host disease, organ
transplant rejection,
acute or chronic immune disease associated with organ transplantation,
sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease,
Grave's
disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's
granulomatosis, Henoch-
Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active
hepatitis, uveitis,
septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious
diseases, parasitic
diseases, acute transverse myelitis, Huntington's chorea, Parkinson's disease,
Alzheimer's
disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies,
heart failure,
myocardial infarction, Addison's disease, sporadic, polyglandular deficiency
type I and
polyglandular deficiency type II, Schmidt's syndrome, adult respiratory
distress syndrome,
alopecia, alopecia greata, seronegative arthopathy, arthropathy, Reiter's
disease, psoriatic
arthropathy, ulcerative colitic arthropathy, enteropathic synovitis,
chlamydia, yersinia and
salmonella associated arthropathy, spondyloarthopathy, atheromatous disease1
arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus
vulgaris, pemphigus
foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anemia,
Coombs positive
haemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia,
myalgic
encephalitis/Royal Free Disease. chronic mucocutaneous candidiasis, giant cell
arteritis,
primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency
Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis C,
common
varied immunodeficiency, common variable hypogammaglobulinemia, dilated
cardiomyopathy, female infertility, ovarian failure, premature ovarian
failure, fibrotic lung
disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung
disease, interstitial
pneumonitis, connective tissue disease associated interstitial lung disease,
mixed connective
tissue disease associated lung disease, systemic sclerosis associated
interstitial lung
disease, rheumatoid arthritis associated interstitial lung disease, systemic
lupus
erythematosus associated lung disease, dermatomyositis/polymyositis associated
lung
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disease, Sjogren's disease associated lung disease, ankylosing spondylitis
associated lung
disease, vasculitic diffuse lung disease, haemosiderosis associated lung
disease, drug-
induced interstitial lung disease, radiation fibrosis, bronchiolitis
obliterans, chronic eosinophilic
pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial
lung disease, gouty
arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis, classical
autoimmune or lupoid
hepatitis, type-2 autoimmune hepatitis, anti-LKM antibody hepatitis,
autoimmune mediated
hypoglycemia, type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, acute
immune disease associated with organ transplantation, chronic immune disease
associated
with organ transplantation, osteoarthrosis, primary sclerosing cholangitis,
idiopathic
leucopaenia, autoimmune neutropenia, renal disease NOS, glomerulonephritides,
microscopic vasulitis of the kidneys, Lyme disease, discoid lupus
erythematosus, male
infertility idiopathic or NOS, sperm autoimmunity, all subtypes of multiple
sclerosis,
sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue
disease,
Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute
rheumatic
fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjogren's
syndrome,
Takayasu's disease/arteritis, autoimmune thrombocytopenia, idiopathic
thrombocytopenia,
autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune
hypothyroidism or
Hashimoto's disease, atrophic autoimmune hypothyroidism, primary myxoedema,
phacogenic
uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver
diseases, allergy and
asthma, mental disorders, depression, schizophrenia, Th2 Type and Thl Type
mediated
diseases, Chronic Obstructive Pulmonary Disease (COPD), inflammatory,
autoimmune and
bone diseases.
[0107] The present antibodies and fragments can also be used to treat or
prevent IL-113
related, or IL-18 related, or autoinflammatory, or autoimmune or inflammation
or immune
diseases.
[0108] Though the genesis of these diseases often involved multistep pathways
and
often multiple different biological systems/pathways, intervention at critical
points in one or
more of these pathways can have an ameliorative or therapeutic effect.
Therapeutic
intervention can occur by either antagonism of a detrimental process/pathway
or stimulation
of a beneficial process/pathway.
[0109] T lymphocytes (T cells) are an important component of a mammalian
immune
response. T cells recognize antigens which are associated with a self-molecule
encoded by
genes within the major histocompatibility complex (MHC). The antigen may be
displayed
together with MHC molecules on the surface of antigen presenting cells, virus
infected cells,
cancer cells, grafts, etc. The T cell system eliminates these altered cells
which pose a health
threat to the host animal. T cells include helper T cells and cytotoxic T
cells. Helper T cells
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proliferate extensively following recognition of an antigen-MHC complex on an
antigen
presenting cell. Helper T cells also secrete a variety of cytokines, e.g.,
lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells and a
variety of other cells
which participate in the immune response.
[0110] In many immune responses, inflammatory cells infiltrate the site of
injury or
infection. The migrating cells may be neutrophilic, eosinophilic, monocytic or
lymphocytic as
can be determined by histologic examination of the affected tissues. See,
e.g., Current
Protocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.
Many immune
related diseases are known and have been extensively studied. Such diseases
include
immune-mediated inflammatory diseases (e.g., rheumatoid arthritis, immune
mediated renal
disease, hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis,
and asthma),
non-immune-mediated inflammatory diseases, infectious diseases,
immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of immunology,
targets were
identified for the treatment of inflammation and inflammatory disorders. In
the area of
immunology, targets have been identified herein for the treatment of
inflammation and
inflammatory disorders. Immune related diseases, in one instance, could be
treated by
suppressing the immune response. Using neutralizing antibodies that inhibit
molecules
having immune stimulatory activity would be beneficial in the treatment of
immune-mediated
and inflammatory diseases. Molecules which inhibit the immune response can be
utilized
(proteins directly or via the use of antibody agonists) to inhibit the immune
response and thus
ameliorate immune related disease.
[0111] As used herein, the term "immunoadhesin" designates molecules which
combine
the binding specificity of a heterologous protein (an "adhesin") with the
effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins comprise a
fusion of an
amino acid sequence with a desired binding specificity, which amino acid
sequence is other
than the antigen recognition and binding site of an antibody (i.e., is
"heterologous"), and an
Fc region (e.g., CH2 and/or CH3 sequence of an IgG). Exemplary adhesin
sequences
include contiguous amino acid sequences that comprise a portion of a receptor
(e.g.,
extracellular domain) or a ligand that binds to a protein of interest. Adhesin
sequences can
also be sequences that bind a protein of interest, but are not receptor or
ligand sequences
(e.g., adhesin sequences in peptibodies). Such polypeptide sequences can be
selected or
identified by various methods, include phage display techniques and high
throughput sorting
methods. The immunoglobulin constant domain sequence in the immunoadhesin can
be
obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4
subtypes, IgA
(including IgA-1 and IgA-2), IgE, IgD, or IgM. Exemplary molecules are the
bispecific CD4-
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IgG molecules described in Berg et al., 1991, PNAS (USA) 88:4723-and Chamow et
al.,
1994, J. Immunol. 153:4268.
[0112] An "isolated" antibody is one that has been identified and separated
and/or
recovered from a component of its natural environment. Contaminant components
of its
natural environment are materials that would interfere with diagnostic or
therapeutic uses for
the antibody, and may include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In some embodiments, the antibody will be purified
(1) to greater
than 95% by weight of antibody as determined by the Lowry method, and most
preferably
more than 99% by weight, (2) to a degree sufficient to obtain at least 15
residues of N-
terminal or internal amino acid sequence by use of a spinning cup sequenator,
or (3) to
homogeneity by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue
or, preferably, silver stain. Isolated antibody includes the antibody in situ
within recombinant
cells since at least one component of the antibody's natural environment will
not be present.
Ordinarily, however, isolated antibody will be prepared by at least one
purification step.
[0113] An "isolated" nucleic acid molecule is a nucleic acid molecule that is
identified and
separated from at least one contaminant nucleic acid molecule with which it is
ordinarily
associated in the natural source of the antibody nucleic acid. An isolated
nucleic acid
molecule is other than in the form or setting in which it is found in nature.
Isolated nucleic acid
molecules therefore are distinguished from the nucleic acid molecule as it
exists in natural
cells. However, an isolated nucleic acid molecule includes a nucleic acid
molecule contained
in cells that ordinarily express the antibody where, for example, the nucleic
acid molecule is in
a chromosomal location different from that of natural cells.
[0114] The term "knob-into-hole" or "KnH" as mentioned herein refers to the
technology
directing the selectively pairing of two polypeptides together in vitro or in
vivo by introducing a
pertuberance (knob) into one polypeptide and a cavity (hole) into the other
polypeptide at an
interface in which they interact. For example, KnHs have been introduced in
the Fc:Fc
binding interfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies (e.g.,
US20007/0178552, WO 96/027011, WO 98/050431and Zhu et al. (1997) Protein
Science
6:781-788). This is especially useful in driving the pairing of two different
heavy chains
together during the manufacture of multispecific antibodies. For example,
multispecific
antibodies having KnH in their Fc regions can further comprise single variable
domains linked
to each Fc region, or further comprise different heavy chain variable domains
that pair with
similar or different light chain variable domains. In fact, KnH technology can
be used to pair
two different receptor extracellular domains together or any other polypeptide
sequences that
comprises different target recognition sequences (e.g., including affibodies,
peptibodies and
other Fc fusions).
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[0115] The expression "linear antibodies" generally refers to the antibodies
described in
Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies
comprise a pair
of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light
chain
polypeptides, form a pair of antigen binding regions. Linear antibodies can be
bispecific or
monospecific.
[0116] The term "mammal" includes any animals classified as mammals, including
humans, cows, horses, dogs, and cats. In one embodiment the mammal is a human.
[0117] The term "monoclonal antibody" as used herein refers to an antibody
obtained
from a population of substantially homogeneous antibodies, i.e., the
individual antibodies
comprising the population are identical except for possible naturally
occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly specific,
being directed
against a single antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody
preparations that typically include different antibodies directed against
different determinants
(epitopes), each monoclonal antibody is directed against a single determinant
on the antigen.
The modifier "monoclonal" indicates the character of the antibody as being
obtained from a
substantially homogeneous population of antibodies, and is not to be construed
as requiring
production of the antibody by any particular method. For example, the
monoclonal antibodies
to be used in accordance with the present invention may be made by the
hybridoma method
first described by Kohler et al., 1975, Nature 256:495, or may be made by
recombinant DNA
methods (see, for example, U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also
be isolated from phage antibody libraries using the techniques described in
Clackson et al.,
1991, Nature 352:624-628 and Marks et al., 1991, J. MoL Biol. 222:581-597, for
example.
[0118] The monoclonal antibodies herein specifically include "chimeric"
antibodies
(immunoglobulins) in which a portion of the heavy and/or light chain is
identical with or
homologous to corresponding sequences in antibodies derived from a particular
species or
belonging to a particular antibody class or subclass, while the remainder of
the chain(s) is
identical with or homologous to corresponding sequences in antibodies derived
from another
species or belonging to another antibody class or subclass, as well as
fragments of such
antibodies, so long as they exhibit the desired biological activity (U.S. Pat.
No. 4,816,567; and
Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855).
[0119] The term "multispecific antibody" is used in the broadest sense and
refers to an
antibody that has polyeptopic specificity. Such multispecific antibodies
include, but are not
limited to, an antibody comprising a heavy chain variable domain (VH) and a
light chain
variable domain (VL), where the VHVL unit has polyepitopic specificity,
antibodies having two
or more VI_ and VH domains with each VHVL unit binding to a different epitope,
antibodies
having two or more single variable domains with at least two single variable
domains binding
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to different epitopes, full length antibodies, antibody fragments such as Fab,
Fv, dsFv, scFv,
diabodies,tandem antibodies, linear antibodies and triabodies, antibody
fragments that have
been linked covalently or bind to each other through non-covalent
interactions. Other
examples of antibody formats have been used or may be used to create
multispecific
antibodies include, but are not limited to, Fc fusions of diabodies, tandem
antibodies, and
single chain antibodies (e.g, Db-Fc, taDb-Fc, taDb-CH3 and (scFV)4-Fc), knob-N-
hole (KnH)
antibodies, octopus antibodies and DAF antibodies.
[0120] "Multispecific Molecule" as used herein refers to a molecule that has
polyepitopic
specificity. "Polyepitopic specificity" refers to the ability to specifically
bind to \at least two
different epitopes on one target molecule or on a different target molecules.
"Monospecific"
refers to the ability to bind only one epitope. According to one embodiment a
multispecific
molecule binds to each epitope with an affinity of 5pM to 0.001pM, 3pM to
0.001pM, 1pM to
0.001pM, 0.5pM to 0.001pM or 0.1pM to 0.001pM. The term "bispecific" as used
herein
refers to the ability to bind two epitopes (e.g, an anti-IL-18/IL-18
bispecific antibody).
Examples of molecules that support or can be engineered to support
polyepitopic specificity
include, but is not limited to, antibodies, affibodies, immunoadhesins,
peptibodies and other
Fc fusions.
[0121] The term "octopus" antibody or antibodies as used herein refers to
multivalent
antibodies comprising an Fc region and two or more antigen binding sites amino-
terminal to
the Fc region (e.g., W001/77342, Wu et al. (2007) Nature Biotechnology, and WO
2007/024715). In one preferred embodiment, the configuration of a polypeptide
of the
antibody is VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,
VD2 is a second
variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2
represent an amino
acid or polypeptide, and n is 0 or 1. In one embodiment, X1 or X2 is a CH1
domain, a portion
of a CH1 domain, some other linker sequence such as a GS linker or some
combination
thereof (e.g., page 5 of WO 2007/024715).
[0122] A nucleic acid is "operably linked," as used herein, when it is placed
into a
functional relationship with another nucleic acid sequence. For example, DNA
for a
presequence or secretory leader is operably linked to DNA for a antibody if it
is expressed as
a preprotein that participates in the secretion of the antibody; 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, an enhancer may not have to be contiguous. Linking is accomplished by
ligation at
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convenient restriction sites. If such sites do not exist, synthetic
oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
[0123] "Peptibody" or "peptibodies" refers to a fusion of peptide sequences
with an Fc
domain. See U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feige et al.
(incorporated by
reference in its entirety). They include one or more peptides linked to the N-
terminus, C-
terminus, amino acid sidechains, or to more than one of these sites. Peptibody
technology
enables design of therapeutic agents that incorporate peptides that target one
or more
ligands or receptors, tumor-homing peptides, membrane-transporting peptides,
and the like.
Peptibody technology has proven useful in design of a number of such
molecules, including
linear and disulfide-constrained peptides, "tandem peptide multimers" (i.e.,
more than one
peptide on a single chain of an Fc domain). See, for example, U.S. Pat. No.
6,660,843; U.S.
Pat. App. No. 2003/0195156, published Oct. 16, 2003 (corresponding to WO
02/092620,
published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18,
2003
(corresponding to WO 03/031589, published Apr. 17, 2003); U.S. Ser. No.
09/422,838, filed
Oct. 22, 1999 (corresponding to WO 00/24770, published May 4, 2000); U.S. Pat.
App. No.
2003/0229023, published Dec. 11,2003; WO 03/057134, published Jul. 17, 2003;
U.S. Pat.
App. No. 2003/0236193, published Dec. 25, 2003 (corresponding to
PCT/U504/010989, filed
Apr. 8, 2004); U.S. Ser. No. 10/666,480, filed Sep. 18, 2003 (corresponding to
WO
04/026329, published Apr. 1, 2004), each of which is hereby incorporated by
reference in its
entirety.
[0124] For the purposes herein, a "pharmaceutical composition" is one that is
adapted
and suitable for administration to a mammal, especially a human. Thus, the
composition can
be used to treat a disease or disorder in the mammal. Moreover, the protein in
the
composition has been subjected to one or more purification or isolation steps,
such that
contaminant(s) that might interfere with its therapeutic use have been
separated therefrom.
Generally, the pharmaceutical composition comprises the therapeutic protein
and a
pharmaceutically acceptable carrier or diluent. The composition is usually
sterile and may be
lyophilized. Pharmaceutical preparations are described in more detail below.
[0125] "Polynucleotide," or "nucleic acid," as used interchangeably herein,
refer to
polymers of nucleotides of any length, and include DNA and RNA. The
nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or
their analogs, or
any substrate that can be incorporated into a polymer by DNA or RNA polymerase
or by a
synthetic reaction. A polynucleotide may comprise modified nucleotides, such
as methylated
nucleotides and their analogs. If present, modification to the nucleotide
structure may be
imparted before or after assembly of the polymer. The sequence of nucleotides
may be
interrupted by non-nucleotide components. A polynucleotide may be further
modified after
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synthesis, such as by conjugation with a label. Other types of modifications
include, for
example, "caps", substitution of one or more of the naturally occurring
nucleotides with an
analog, internucleotide modifications such as, for example, those with
uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,
etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those
containing
pendant moieties, such as, for example, proteins (e.g., nucleases, toxins,
antibodies, signal
peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron, oxidative
metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha anomeric
nucleic acids, etc.),
as well as unmodified forms of the polynucleotide(s). Further, any of the
hydroxyl groups
ordinarily present in the sugars may be replaced, for example, by phosphonate
groups,
phosphate groups, protected by standard protecting groups, or activated to
prepare additional
linkages to additional nucleotides, or may be conjugated to solid or semi-
solid supports. The
5' and 3' terminal OH can be phosphorylated or substituted with amines or
organic capping
group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be
derivatized to
standard protecting groups. Polynucleotides can also contain analogous forms
of ribose or
deoxyribose sugars that are generally known in the art, including, for
example, 2'-0-methyl-,
2'-0-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-
anomeric sugars,
epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars,
furanose sugars,
sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl
riboside.
One or more phosphodiester linkage may be replaced by alternative linking
groups. These
alternative linking groups include, but are not limited to, embodiments
wherein phosphate is
replaced by P(0)S("thioate"), P(S)S ("dithioate"), "(0)NR2 ("amidate"), P(0)R,
P(0)OR', CO
or CH2 ("formacetal"), in which each R or R' is independently H or substituted
or
unsubstituted alkyl (1-20 C.) optionally containing an ether (-0¨) linkage,
aryl, alkenyl,
cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide
need be identical. The
preceding description applies to all polynucleotides referred to herein,
including RNA and
DNA.
[0126] "Oligonucleotide," as used herein, generally refers to short, generally
single
stranded, generally synthetic polynucleotides that are generally, but not
necessarily, less than
about 200 nucleotides in length. The terms "oligonucleotide" and
"polynucleotide" are not
mutually exclusive. The description above for polynucleotides is equally and
fully applicable to
oligonucleotides.
[0127] The term "receptor binding domain" is used to designate any native
ligand for a
receptor, including cell adhesion molecules, or any region or derivative of
such native ligand
retaining at least a qualitative receptor binding ability of a corresponding
native ligand. This
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definition, among others, specifically includes binding sequences from ligands
for the above-
mentioned receptors.
[0128] "Secretion signal sequence" or "signal sequence" refers to a nucleic
acid
sequence encoding a short signal peptide that can be used to direct a newly
synthesized
protein of interest through a cellular membrane, usually the inner membrane or
both inner and
outer membranes of prokaryotes. As such, the protein of interest such as the
immunoglobulin
light or heavy chain polypeptide is secreted into the periplasm of the
prokaryotic host cells or
into the culture medium. The signal peptide encoded by the secretion signal
sequence may
be endogenous to the host cells, or they may be exogenous, including signal
peptides native
to the polypeptide to be expressed. Secretion signal sequences are typically
present at the
amino terminus of a polypeptide to be expressed, and are typically removed
enzymatically
between biosynthesis and secretion of the polypeptide from the cytoplasm.
Thus, the signal
peptide is usually not present in a mature protein product.
[0129] The expression "single domain antibodies" (sdAbs) or "single variable
domain
(SVD) antibodies" generally refers to antibodies in which a single variable
domain (VH or VL)
can confer antigen binding. In other words, the single variable domain need
not interact with
another variable domain in order to bind the target antigen. Examples of
single domain
antibodies include, but is not limited to, those derived from nature such as
camelids (lamas
and camels) and cartilaginous fish (e.g., nurse sharks) and those derived from
recombinant
methods from humans and mouse antibodies (Nature (1989) 341:544-546; Dev Comp
Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends
Biotechnol
(2003):21:484-490; WO 2005/035572; WO 03/035694; Febs Lett (1994) 339:285-290;
W000/29004; WO 02/051870).
[0130] As used herein, a "therapeutic antibody" is an antibody that is
effective in treating
a disease or disorder in a mammal with or predisposed to the disease or
disorder. Exemplary
therapeutic antibodies include the anti-IL-1 13 and anti-IL-18 antibodies of
the present
invention, including the anti-IL-113 and anti-IL-18 bispecific antibodies of
the present invention,
as well as antibodies including rhuMAb 4D5 (HERCEPTINO) (Carter et al., 1992,
Proc. Natl.
Acad. Sci. USA, 89:4285-4289, U.S. Pat. No. 5,725,856); anti-CD20 antibodies
such as
chimeric anti-CD20 "C2B8" as in U.S. Pat. No. 5,736,137 (RITUXANO), a chimeric
or
humanized variant of the 2H7 antibody as in U.S. Pat. No. 5,721,108, B1 or
Tositumomab
(BEXXARO); anti-IL-8 (St John et al., 1993, Chest, 103:932, and International
Publication No.
WO 95/23865); anti-VEGF antibodies including humanized and/or affinity matured
anti-VEGF
antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN TM (Kim
et al.,
1992, Growth Factors, 7:53-64, International Publication No. WO 96/30046, and
WO
98/45331, published Oct. 15, 1998); anti-PSCA antibodies (W001/40309); anti-
CD40
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antibodies, including S2C6 and humanized variants thereof (W000/75348); anti-
CD 11a (U.S.
Pat. No. 5,622,700, WO 98/23761, Steppe et al, 1991, Transplant Intl. 4:3-7,
and Hourmant
et al., 1994, Transplantation 58:377-380); anti-IgE (Presta et al., 1993, J.
Immunol. 151:2623-
2632, and International Publication No. WO 95/19181); anti-CD18 (U.S. Pat. No.
5,622,700,
issued Apr. 22, 1997, or as in WO 97/26912, published Jul. 31, 1997); anti-IgE
(U.S. Pat. No.
5,714,338, issued Feb. 3, 1998 or U.S. Pat. No. 5,091,313, issued Feb. 25,
1992, WO
93/04173 published Mar. 4, 1993, or International Application No.
PCT/US98/13410 filed Jun.
30, 1998, U.S. Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793
published
Nov. 19, 1998); anti-TNF-a antibodies including cA2 (REMICADEO), CDP571 and
MAK-195
(See, U.S. Pat. No. 5,672,347 issued Sep. 30, 1997, Lorenz et al. 1996, J.
Immunol.
156(4):1646-1653, and Dhainaut et al. 1995, Crit. Care Med. 23(9):1461-1469);
anti-Tissue
Factor (TF) (European Patent No. 0 420 937 B1 granted Nov. 9, 1994); anti-
human y4137
integrin (WO 98/06248 published Feb. 19, 1998); anti-EGFR (chimerized or
humanized 225
antibody as in WO 96/40210 published Dec. 19, 1996); anti-CD3 antibodies such
as OKT3
(U.S. Pat. No. 4,515,893 issued May 7, 1985); anti-CD25 or anti-tac antibodies
such as CHI-
621 (SIMULECTO) and (ZENAPAX0) (See U.S. Pat. No. 5,693,762 issued Dec. 2,
1997);
anti-CD4 antibodies such as the cM-7412 antibody (Choy et al. 1996, Arthritis
Rheum
39(1):52-56); anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al. 1988,
Nature
332:323-337; anti-Fc receptor antibodies such as the M22 antibody directed
against FcyRI as
in Graziano et al. 1995, J. Immunol. 155(10):4996-5002; anti-carcinoembryonic
antigen (CEA)
antibodies such as hMN-14 (Sharkey et al. 1995, Cancer Res. 55(23 Suppl):
5935s-5945s;
antibodies directed against breast epithelial cells including huBrE-3, hu-Mc 3
and CHL6
(Ceriani et al. 1995, Cancer Res. 55(23): 5852s-5856s; and Richman et al.
1995, Cancer
Res. 55(23 Supp): 5916s-5920s); antibodies that bind to colon carcinoma cells
such as C242
(Litton et al. 1996, Eur J. Immunol. 26(1): 1-9); anti-CD38 antibodies, e.g.
AT 13/5 (Ellis et al.
1995, J. Immunol. 155(2):925-937); anti-CD33 antibodies such as Hu M195
(Jurcic et al.
1995, Cancer Res 55(23 Suppl):5908s-5910s and CMA-676 or CDP771; anti-CD22
antibodies such as LL2 or LymphoCide (Juweid et al. 1995, Cancer Res 55(23
Suppl):5899s-
5907s; anti-EpCAM antibodies such as 17-1A (PANOREX0); anti-Gpl lb/Illa
antibodies such
as abciximab or c7E3 Fab (REOPROO); anti-RSV antibodies such as MEDI-493
(SYNAGISO); anti-CMV antibodies such as PROTOVIRO; anti-HIV antibodies such as
PR0542; anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIRO;
anti-CA 125
antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2; anti-avp antibody
VITAXINO;
anti-human renal cell carcinoma antibody such as ch-G250; ING-1; anti-human 17-
IA
antibody (3622W94); anti-human colorectal tumor antibody (A33); anti-human
melanoma
antibody R24 directed against GD3 ganglioside; anti-human squamous-cell
carcinoma (SF-
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WO 2012/021773 CA 02808185 2013-02-12PCT/US2011/047532
25); and anti-human leukocyte antigen (HLA) antibodies such as Smart ID10 and
the anti-
HLA DR antibody Oncolym (Lym-1).
[0131] "Target molecule" refers to a molecule that is capable of binding a
target
recognition site. Examples of target molecule:target recognition site
interactions include
antigen:antibody variable domain interactions, receptorligand interactions,
ligand:receptor
interactions, adhesin:adhesin interactions, biotin:strepavidin interactions,
etc. In one
embodiment, the target molecule is a biological molecule.
[0132] The term "therapeutically effective amount" refers to an amount of a
composition
of this invention effective to "alleviate" or "treat" a disease or disorder in
a subject or mammal.
In one embodiment, "therapeutically effective amount" is intended to include
an amount of the
antibodies described herein alone or in combination with other active
ingredients effective to
inhibit or decrease IL-1beta and IL-18 binding to their receptors or effective
to treat or prevent
inflammatory disorders in a subject in need thereof.
[0133] "Treatment" (and grammatical variations thereof such as "treat" or
"treating")
refers to clinical intervention in an attempt to alter the natural course of
the subject being
treated, and can be performed either for prophylaxis or during the course of
clinical pathology.
Desirable effects of treatment include, but are not limited to, preventing
occurrence or
recurrence of disease, alleviation of symptoms, diminishment of any direct or
indirect
pathological consequences of the disease, preventing metastasis, decreasing
the rate of
disease progression, amelioration or palliation of the disease state, and
remission or
improved prognosis. In some embodiments, antibodies of the invention are used
to delay
development of a disease or to slow the progression of a disease. Generally,
treatment of a
disease or disorder involves the lessening of one or more symptoms or medical
problems
associated with the disease or disorder. In some embodiments, antibodies and
compositions
of this invention can be used to prevent the onset or reoccurrence of the
disease or disorder
in a subject or mammal. For example, in a subject with autoimmune disease, an
antibody of
this invention can be used to prevent or treat flare-ups. Consecutive
treatment or
administration refers to treatment on at least a daily basis without
interruption in treatment by
one or more days. Intermittent treatment or administration, or, treatment or
administration in
an intermittent fashion, refers to treatment that is not consecutive, but
rather cyclic in nature.
The treatment regime herein may be either consecutive or intermittent.
[0134] The term "variable" refers to the fact that certain segments of the
variable
domains differ extensively in sequence among antibodies. The V domain mediates
antigen
binding and defines specificity of a particular antibody for its particular
antigen. However, the
variability is not evenly distributed across the amino acid span of a variable
domain. Instead,
the V region consist of relatively invariant stretches called framework
regions (FRs) of
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WO 2012/021773 CA 02808185 2013-02-12PCT/US2011/047532
separated by shorter regions of extreme variability called "hypervariable
regions". The
hypervariable regions in one variable domain may cooperate with the
hypervariable regions
from another chain to contribute to the formation of a antigen-binding site on
antibodies,
depending on the type of antibody (see Kabat et al., Sequences of Proteins of
Immunological
Interest, 5th Ed. Public Health Service, National Institutes of Health,
Bethesda, MD (1991)).
Constant domains are not typically involved directly in binding an antibody to
an antigen, but
exhibit various effector functions, such as participation of the antibody in
antibody dependent
cellular cytotoxicity (ADCC).
[0135] A "variant" or "altered" heavy chain, as used herein, generally refers
to a heavy
chain with reduced disulfide linkage capability, for e.g., wherein at least
one cysteine residue
has been rendered incapable of disulfide linkage formation. Preferably, said
at least one
cysteine is in the hinge region of the heavy chain.
[0136] The term "vector," as used herein, is intended to refer to a nucleic
acid molecule
capable of transporting another nucleic acid to which it has been linked. One
type of vector is
a "plasmid", a circular double stranded DNA loop into which additional DNA
segments may be
ligated. Another type of vector is a phage vector. Another type of vector is a
viral vector,
wherein additional DNA segments may be ligated into the viral genome. Certain
vectors are
capable of autonomous replication in a host cell into which they are
introduced (for example,
bacterial vectors having a bacterial origin of replication and episomal
mammalian vectors).
Other vectors (for example, non-episomal mammalian vectors) can be integrated
into the
genome of a host cell upon introduction into the host cell, and thereby are
replicated along
with the host genome. Moreover, certain vectors are capable of directing the
expression of
genes to which they are operatively linked. Such vectors are referred to
herein as
"recombinant expression vectors" (or simply, "recombinant vectors"). In
general, expression
vectors of utility in recombinant DNA techniques are often in the form of
plasmids. In the
present specification, "plasmid" and "vector" may be used interchangeably as
the plasmid is
the most commonly used form of vector.
[0137] An antibody that "selectively binds" a target molecule with
significantly better
affinity than it binds to other molecules that are not the target molecule.
The relative binding
and/or binding affinity may be demonstrated in a variety of methods accepted
in the art
including, but not limited to: enzyme linked immunosorbent assay (ELISA) and
fluorescence
activated cell sorting (FACS). In some embodiments, the antibody of the
invention binds a
target molecule with at least about 1 log higher concentration reactivity than
it binds to a non-
target molecule, as determined by an ELISA.
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I. Exemplary Antibodies
[0138] Soluble human IL-113 or human IL-18, or fragments thereof, optionally
conjugated
to other molecules, can be used as immunogens for generating antibodies.
Alternatively, or
additionally, cells expressing human IL-113 or human IL-18 can be used as the
immunogen.
Such cells can be derived from a natural source or may be cells that have been
transformed
by recombinant techniques to express human IL-1[3 or human IL-18. Other forms
of human
IL-113 or human IL-18 useful for preparing antibodies will be apparent to
those in the art.
A. Polyclonal Antibodies
[0139] Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous
(sc) or intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful
to conjugate the relevant antigen to a protein that is immunogenic in the
species to be
immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing agent, for
example,
maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine
residues), N-
hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic
anhydride, SOCl2, or
RiN=C=NR, where R and R1 aredifferent alkyl groups.
[0140] Animals are immunized against the antigen, immunogenic conjugates, or
derivatives by combining, for example, 100 pg or 5 pg of the protein or
conjugate (for rabbits
or mice, respectively) with 3 volumes of Freund's complete adjuvant and
injecting the solution
intradermally at multiple sites. Approximately one month later, the animals
are boosted with
1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete
adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later the animals
are bled and the
serum is assayed for antibody titer. Animals are boosted until the titer
plateaus. Preferably,
the animal is boosted with the conjugate of the same antigen, but conjugated
to a different
protein and/or through a different cross-linking reagent. Conjugates also can
be made in
recombinant cell culture as protein fusions. Also, aggregating agents such as
alum are
suitably used to enhance the immune response.
B. Monoclonal Antibodies
[0141] Monoclonal antibodies may be made using the hybridoma method first
described
by Kohler et al., 1975, Nature, 256:495, or may be made by recombinant DNA
methods (See,
for example, U.S. Pat. No. 4,816,567).
[0142] In the hybridoma method, a mouse or other appropriate host animal, such
as a
hamster or macaque monkey, is immunized as hereinabove described to elicit
lymphocytes
that produce or are capable of producing antibodies that will specifically
bind to the protein
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used for immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes
then are fused with myeloma cells using a suitable fusing agent, such as
polyethylene glycol,
to form a hybridoma cell (Goding, 1986, Monoclonal Antibodies: Principles and
Practice, pp.
59-103 (Academic Press)).
[0143] The hybridoma cells thus prepared are seeded and grown in a suitable
culture
medium that preferably contains one or more substances that inhibit the growth
or survival of
the unfused, parental myeloma cells. For example, if the parental myeloma
cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the
culture
medium for the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine
(HAT medium), which substances prevent the growth of HGPRT-deficient cells.
[0144] Preferred myeloma cells are those that fuse efficiently, support stable
high-level
production of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. Among these, preferred myeloma cell lines are
murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors
available
from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and
SP-2 or X63-Ag8-
653 cells available from the American Type Culture Collection, Rockville, Md.
USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been described for
the
production of human monoclonal antibodies (Kozbor, 1984, J. Immunol.,
133:3001; Brodeur
et al., 1987, Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel
Dekker, Inc., New York)).
[0145] Culture medium in which hybridoma cells are growing is assayed for
production of
monoclonal antibodies directed against the antigen. Preferably, the binding
specificity of
monoclonal antibodies produced by hybridoma cells is determined by
immunoprecipitation or
by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA).
[0146] After hybridoma cells are identified that produce antibodies of the
desired
specificity, affinity, and/or activity, the clones may be subcloned by
limiting dilution procedures
and grown by standard methods (Goding, supra). Suitable culture media for this
purpose
include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma
cells may be
grown in vivo as ascites tumors in an animal.
[0147] The monoclonal antibodies secreted by the subclones are suitably
separated from
the culture medium, ascites fluid, or serum by conventional immunoglobulin
purification
procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0148] DNA encoding the monoclonal antibodies is readily isolated and
sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding
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WO 2012/021773 CA 02808185 2013-02-12PCT/US2011/047532
specifically to genes encoding the heavy and light chains of the monoclonal
antibodies). The
hybridoma cells serve as a preferred source of such DNA. Once isolated, the
DNA may be
placed into expression vectors, which are then transfected into host cells
such as E. coli cells,
simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise
produce immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the
recombinant host cells. Recombinant production of antibodies will be described
in more detail
below.
[0149] In a further embodiment, antibodies or antibody fragments can be
isolated from
antibody phage libraries generated using the techniques described in
McCafferty et al., 1990,
Nature, 348:552-554. Clackson et al., 1991, Nature, 352:624-628, and Marks et
al., 1991, J.
Mol. Biol., 222:581-597 describe the isolation of murine and human antibodies,
respectively,
using phage libraries. Subsequent publications describe the production of high
affinity (nM
range) human antibodies by chain shuffling (Marks et al., 1992,
Bio/Technology, 10:779-783),
as well as combinatorial infection and in vivo recombination as a strategy for
constructing very
large phage libraries (Waterhouse et al., 1993, Nuc. Acids. Res., 21:2265-
2266). Thus, these
techniques are viable alternatives to traditional monoclonal antibody
hybridoma techniques for
isolation of monoclonal antibodies.
[0150] The DNA also may be modified, for example, by substituting the coding
sequence
for human heavy- and light-chain constant domains in place of the homologous
murine
sequences (U.S. Pat. No. 4,816,567; Morrison, et al., 1984, Proc. Natl. Acad.
Sci. USA,
81:6851), or by covalently joining to the immunoglobulin coding sequence all
or part of the
coding sequence for non-immunoglobulin material (e.g., protein domains).
[0151] Typically such non-immunoglobulin material is substituted for the
constant
domains of an antibody, or is substituted for the variable domains of one
antigen-combining
site of an antibody to create a chimeric bivalent antibody comprising one
antigen-combining
site having specificity for an antigen and another antigen-combining site
having specificity for
a different antigen.
C. Humanized and Human Antibodies
[0152] A humanized antibody has one or more amino acid residues from a source
that is
non-human. The non-human amino acid residues are often referred to as "import"
residues,
and are typically taken from an "import" variable domain. Humanization can be
performed
generally following the method of Winter and co-workers (Jones et al., 1986,
Nature,
321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al.,
1988, Science,
239:1534-1536), by substituting rodent CDRs or CDR sequences for the
corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies are
chimeric
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antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact
human variable
domain has been substituted by the corresponding sequence from a non-human
species. In
practice, humanized antibodies are typically human antibodies in which some
CDR residues
and possibly some FR residues are substituted by residues from analogous sites
in non-
human, for example, rodent antibodies.
[0153] The choice of human variable domains, both light and heavy, to be used
in
making the humanized antibodies is very important to reduce antigenicity.
According to the
so-called "best-fit" method, the sequence of the variable domain of a rodent
antibody is
screened against the entire library of known human variable-domain sequences.
The human
sequence which is closest to that of the rodent is then accepted as the human
framework
(FR) for the humanized antibody (Sims et al., 1987, J. Immunol., 151:2296;
Chothia et al.,
1987, J. Mol. Biol., 196:901). Another method uses a particular framework
derived from the
consensus sequence of all human antibodies of a particular subgroup of light
or heavy
chains. The same framework may be used for several different humanized
antibodies (Carter
et al., 1992, Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al., 1993, J.
Immunol., 151:2623).
[0154] It is further important that antibodies be humanized with retention of
high affinity
for the antigen and other favorable biological properties. To achieve this
goal, according to a
preferred method, humanized antibodies are prepared by a process of analysis
of the
parental sequences and various conceptual humanized products using three-
dimensional
models of the parental and humanized sequences. Three-dimensional
immunoglobulin
models are commonly available and are familiar to those skilled in the art.
Computer
programs are available which illustrate and display probable three-dimensional
conformational
structures of selected candidate immunoglobulin sequences. Inspection of these
displays
permits analysis of the likely role of the residues in the functioning of the
candidate
immunoglobulin sequence, i.e., the analysis of residues that influence the
ability of the
candidate immunoglobulin to bind its antigen. In this way, FR residues can be
selected and
combined from the recipient and import sequences so that the desired antibody
characteristic,
such as increased affinity for the target antigen(s), is achieved. In general,
the CDR residues
are directly and most substantially involved in influencing antigen binding.
[0155] Alternatively, it is now possible to produce transgenic animals (e.g.,
mice) that are
capable, upon immunization, of producing a full repertoire of human antibodies
in the absence
of endogenous immunoglobulin production. For example, it has been described
that the
homozygous deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and
germ-line mutant mice results in complete inhibition of endogenous antibody
production.
Transfer of the human germ-line immunoglobulin gene array in such germ-line
mutant mice
will result in the production of human antibodies upon antigen challenge. See,
e.g., Jakobovits
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et al., 1993, Proc. Natl. Acad. Sci. USA, 90:2551; Jakobovits et al., 1993,
Nature, 362:255-
258; Bruggermann et al., 1993, Year in Immuno., 7:33; and Duchosal et al.,
1992, Nature
355:258. Human antibodies can also be derived from phage-display libraries
(Hoogenboom et
al., 1991, J. MoL Biol., 227:381; Marks et al., J. Mol. Biol., 1991, 222:581-
597; Vaughan et al.,
1996, Nature Biotech 14:309).
I. Chimeric and Humanized Antibodies
[0156] In certain embodiments, an antibody provided herein is a chimeric
antibody.
Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567;
and Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a
chimeric antibody
comprises a non-human variable region (e.g., a variable region derived from a
mouse, rat,
hamster, rabbit, or non-human primate, such as a monkey) and a human constant
region. In
a further example, a chimeric antibody is a "class switched" antibody in which
the class or
subclass has been changed from that of the parent antibody. Chimeric
antibodies include
antigen-binding fragments thereof.
[0157] In certain embodiments, a chimeric antibody is a humanized antibody.
Typically,
a non-human antibody is humanized to reduce immunogenicity to humans, while
retaining the
specificity and affinity of the parental non-human antibody. Generally, a
humanized antibody
comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions
thereof) are
derived from a non-human antibody, and FRs (or portions thereof) are derived
from human
antibody sequences. A humanized antibody optionally will also comprise at
least a portion of
a human constant region. In some embodiments, some FR residues in a humanized
antibody
are substituted with corresponding residues from a non-human antibody (e.g.,
the antibody
from which the HVR residues are derived), e.g., to restore or improve antibody
specificity or
affinity.
[0158] Humanized antibodies and methods of making them are reviewed, e.g., in
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further
described, e.g., in
Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA
86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and
7,087,409;
Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting);
Padlan, Mo/.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al.,
Methods 36:43-60
(2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68
(2005) and Klimka
et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR
shuffling).
[0159] Human framework regions that may be used for humanization include but
are not
limited to: framework regions selected using the "best-fit" method (see, e.g.,
Sims et al. J.
Immunol. 151:2296 (1993)); framework regions derived from the consensus
sequence of
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human antibodies of a particular subgroup of light or heavy chain variable
regions (see, e.g.,
Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J.
Immunol.,
151:2623 (1993)); human mature (somatically mutated) framework regions or
human
germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.
13:1619-1633
(2008)); and framework regions derived from screening FR libraries (see, e.g.,
Baca et al., J.
Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-
22618
(1996)).
ii. Human Antibodies
[0160] In certain embodiments, an antibody provided herein is a human
antibody.
Human antibodies can be produced using various techniques known in the art.
Human
antibodies are described generally in van Dijk and van de Winkel, Curr. Opin.
Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
[0161] Human antibodies may be prepared by administering an immunogen to a
transgenic animal that has been modified to produce intact human antibodies or
intact
antibodies with human variable regions in response to antigenic challenge.
Such animals
typically contain all or a portion of the human immunoglobulin loci, which
replace the
endogenous immunoglobulin loci, or which are present extrachromosomally or
integrated
randomly into the animal's chromosomes. In such transgenic mice, the
endogenous
immunoglobulin loci have generally been inactivated. For review of methods for
obtaining
human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-
1125 (2005).
See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing
XENOMOUSETm
technology; U.S. Patent No. 5,770,429 describing HuMABO technology; U.S.
Patent No.
7,041,870 describing K-M MOUSE technology, and U.S. Patent Application
Publication No.
US 2007/0061900, describing VELociMousE technology). Human variable regions
from
intact antibodies generated by such animals may be further modified, e.g., by
combining with
a different human constant region.
[0162] Human antibodies can also be made by hybridoma-based methods. Human
myeloma and mouse-human heteromyeloma cell lines for the production of human
monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol.,
133: 3001
(1984); Brodeur et al., Monoclonal Antibody Production Techniques and
Applications, pp. 51-
63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,
147: 86 (1991).)
Human antibodies generated via human B-cell hybridoma technology are also
described in Li
et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those
described, for example, in U.S. Patent No. 7,189,826 (describing production of
monoclonal
human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,
26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma technology (Trioma
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technology) is also described in Vollmers and Brand lein, Histology and
Histopathology,
20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in
Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0163] Human antibodies may also be generated by isolating Fv clone variable
domain
sequences selected from human-derived phage display libraries. Such variable
domain
sequences may then be combined with a desired human constant domain.
Techniques for
selecting human antibodies from antibody libraries are described below.
D. Multispecific Antibodies
[0164] Multispecific antibodies have binding specificities for at least two
different
antigens. While such molecules normally will only bind two antigens (e.g.,
bispecific
antibodies, BsAbs), antibodies with additional specificities such as
trispecific antibodies are
encompassed by this expression when used herein. Examples of BsAbs include
those with
one antigen binding site directed against IL-1[3 and another antigen binding
site directed
against IL-18. In some embodiments, the BsAbs comprise a first binding
specificity for IL-113
or IL-18 and a second binding specificity for an activating receptor having a
cytoplasmic ITAM
motif. An ITAM motif structure possesses two tyrosines separate by a 9-11
amino acid
spacer. A general consensus sequence is YxxL/I(x)6_8YxxL (Isakov, N., 1997, J.
Leukoc. Biol.,
61:6-16). Exemplary activating receptors include FccRI, FcyRIII, FcyRI,
FcyRIIA, and
FcyRIIC. Other activating receptors include, e.g., CD3, CD2, 0D10, CD161, DAP-
12, KAR,
KARAP, FccRII, Trem-1, Trem-2, CD28, p44, p46, B cell receptor, LMP2A, STAM,
STAM-2,
GPVI, and CD40 (See, e.g., Azzoni, et al., 1998, J. Immunol. 161:3493; Kita,
et al., 1999, J.
Immunol. 162:6901; Merchant, et al., 2000, J. Biol. Chem. 74:9115; Pandey, et
al., 2000, J.
Biol. Chem. 275:38633; Zheng, et al., 2001, J. Biol. Chem. 276:12999; Propst,
et al., 2000, J.
Immunol. 165:2214).
[0165] In one embodiment, a BsAb comprises a first binding specificity for IL-
113 and a
second binding specificity for IL-18. Bispecific antibodies can be prepared as
full length
antibodies or antibody fragments (for example, F(ab')2bispecific antibodies).
Bispecific
antibodies may additionally be prepared as knobs-in-holes or hingeless
antibodies. Bispecific
antibodies are reviewed in Segal et al., 2001, J. Immunol. Methods 248:1-6.
[0166] Methods for making bispecific antibodies are known in the art.
Traditional
production of full length bispecific antibodies is based on the coexpression
of two
immunoglobulin heavy chain-light chain pairs, where the two chains have
different specificities
(Millstein et al., 1983, Nature, 305:537-539). Because of the random
assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a
potential
mixture of 10 different antibody molecules, of which only one has the correct
bispecific
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structure. Purification of the correct molecule, usually done by affinity
chromatography steps,
is rather cumbersome, and the product yields are low. Similar procedures are
disclosed in
WO 93/08829, and in Traunecker et al., 1991, EMBO J., 10:3655-3659.
[0167] According to a different approach, antibody variable domains with the
desired
binding specificities (antibody-antigen combining sites) are fused to
immunoglobulin constant
domain sequences. The fusion can be with an immunoglobulin heavy chain
constant domain,
comprising at least part of the hinge, CH2, and CH3 regions. It is preferred
to have the first
heavy-chain constant region (CH1) containing the site necessary for light
chain binding,
present in at least one of the fusions. DNAs encoding the immunoglobulin heavy
chain
fusions and, if desired, the immunoglobulin light chain, are inserted into
separate expression
vectors, and are co-transfected into a suitable host organism. This provides
for great flexibility
in adjusting the mutual proportions of the three antibody fragments in
embodiments when
unequal ratios of the three antibody chains used in the construction provide
the optimum
yields. It is, however, possible to insert the coding sequences for two or all
three antibody
chains in one expression vector when the expression of at least two antibody
chains in equal
ratios results in high yields or when the ratios are of no particular
significance.
[0168] In another embodiment of this approach, the bispecific antibodies are
composed
of a hybrid immunoglobulin heavy chain with a first binding specificity in one
arm, and a hybrid
immunoglobulin heavy chain-light chain pair (providing a second binding
specificity) in the
other arm. It was found that this asymmetric structure facilitates the
separation of the desired
bispecific compound from unwanted immunoglobulin chain combinations, as the
presence of
an immunoglobulin light chain in only one half of the bispecific molecule
provides for a facile
method of separation. This approach is disclosed in WO 94/04690. For further
details of
methods for generating bispecific antibodies, see, for example, Suresh et al.,
1986, Methods
in Enzymology, 121:210.
[0169] According to another approach described in W096/27011, the interface
between
a pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
that are recovered from recombinant cell culture. The preferred interface
comprises at least a
part of the CH3 domain of an antibody constant domain. In this method, one or
more small
amino acid side chains from the interface of the first antibody molecule are
replaced with
larger side chains (for example, tyrosine or tryptophan). Compensatory
"cavities" of identical
or similar size to the large side chain(s) are created on the interface of the
second antibody
molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or
threonine). This provides a mechanism for increasing the yield of the
heterodimer over other
unwanted end-products such as homodimers.
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[0170] Bispecific antibodies include cross-linked or "heteroconjugate"
antibodies. For
example, one of the antibodies in the heteroconjugate can be coupled to
avidin, the other to
biotin. Such antibodies have, for example, been proposed to target immune
system cells to
unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(WO 91/00360,
WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any
convenient cross-linking methods. Suitable cross-linking agents are well known
in the art, and
are disclosed, for example, in U.S. Pat. No. 4,676,980, along with a number of
cross-linking
techniques.
[0171] Antibodies with more than two valencies are also contemplated. For
example,
trispecific antibodies can be prepared According to Tutt et al., 1991, J.
Immunol. 147: 60.
[0172] Engineered antibodies with three or more functional antigen binding
sites,
including "Octopus antibodies," are also included herein (see, e.g. US
2006/0025576A1).
[0173] The antibody or fragment herein also includes a "Dual Acting FAb" or
"DAF"
comprising an antigen binding site that binds to IL-113 as well as IL-18 (see,
US 2008/0069820, for example).
E. Antibodies with Variant Hinge Regions
[0174] The antibodies of the present invention may also comprise variant heavy
chains,
for example as described in application Ser. No. 10/697,995, filed Oct. 30,
2003. Antibodies
comprising variant heavy chains comprise an alteration of at least one
disulfide-forming
cysteine residue, such that the cysteine residue is incapable of forming a
disulfide linkage. In
one aspect, said cysteine(s) is of the hinge region of the heavy chain (thus,
such a hinge
region is referred to herein as a "variant hinge region" and may additionally
be referred to as
"hingeless").
[0175] In some aspects, such immunoglobulins lack the complete repertoire of
heavy
chain cysteine residues that are normally capable of forming disulfide
linkages, either
intermolecularly (such as between two heavy chains) or intramolecularly (such
as between
two cysteine residues in a single polypeptide chain). Generally and
preferably, the disulfide
linkage formed by the cysteine residue(s) that is altered (i.e., rendered
incapable of forming
disulfide linkages) is one that, when not present in an antibody, does not
result in a
substantial loss of the normal physicochemical and/or biological
characteristics of the
immunoglobulin. Preferably, but not necessarily, the cysteine residue that is
rendered
incapable of forming disulfide linkages is a cysteine of the hinge region of a
heavy chain.
[0176] An antibody with variant heavy chains or variant hinge region is
generally
produced by expressing in a host cell an antibody in which at least one, at
least two, at least
three, at least four, or between two and eleven inter-heavy chain disulfide
linkages are
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eliminated, and recovering said antibody from the host cell. Expression of
said antibody can
be from a polynucleotide encoding an antibody, said antibody comprising a
variant heavy
chain with reduced disulfide linkage capability, followed by recovering said
antibody from the
host cell comprising the polynucleotide. Preferably, said heavy chain
comprises a variant
hinge region of an immunoglobulin heavy chain, wherein at least one cysteine
of said variant
hinge region is rendered incapable of forming a disulfide linkage.
[0177] It is further anticipated that any cysteine in an immunoglobulin heavy
chain can be
rendered incapable of disulfide linkage formation, similarly to the hinge
cysteines described
herein, provided that such alteration does not substantially reduce the
biological function of
the immunoglobulin. For example, IgM and IgE lack a hinge region, but each
contains an
extra heavy chain domain; at least one (in some embodiments, all) of the
cysteines of the
heavy chain can be rendered incapable of disulfide linkage formation in
methods of the
invention so long as it does not substantially reduce the biological function
of the heavy chain
and/or the antibody which comprises the heavy chain.
[0178] Heavy chain hinge cysteines are well known in the art, as described,
for example,
in Kabat, 1991, "Sequences of proteins of immunological interest," supra. As
is known in the
art, the number of hinge cysteines varies depending on the class and subclass
of
immunoglobulin. See, for example, Janeway, 1999, Immunobiology, 4th Ed.,
(Garland
Publishing, NY). For example, in human IgGls, two hinge cysteines are
separated by two
prolines, and these are normally paired with their counterparts on an adjacent
heavy chain in
intermolecular disulfide linkages. Other examples include human IgG2 that
contains 4 hinge
cysteines, IgG3 that contains 11 hinge cysteines, and IgG4 that contains 2
hinge cysteines.
[0179] Accordingly, methods of the invention include expressing in a host cell
an
immunoglobulin heavy chain comprising a variant hinge region, where at least
one cysteine of
the variant hinge region is rendered incapable of forming a disulfide linkage,
allowing the
heavy chain to complex with a light chain to form a biologically active
antibody, and
recovering the antibody from the host cell.
[0180] Alternative embodiments include those where at least 2, 3, or 4
cysteines are
rendered incapable of forming a disulfide linkage; where from about two to
about eleven
cysteines are rendered incapable; and where all the cysteines of the variant
hinge region are
rendered incapable.
[0181] Light chains and heavy chains constituting antibodies of the invention
as produced
according to methods of the invention may be encoded by a single
polynucleotide or by
separate polynucleotides.
[0182] Cysteines normally involved in disulfide linkage formation can be
rendered
incapable of forming disulfide linkages by any of a variety of methods known
in the art, or
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those that would be evident to one skilled in the art in view of the criteria
described herein.
For example, a hinge cysteine can be substituted with another amino acid, such
as serine that
is not capable of disulfide bonding. Amino acid substitution can be achieved
by standard
molecular biology techniques, such as site directed mutagenesis of the nucleic
acid sequence
encoding the hinge region that is to be modified. Suitable techniques include
those described
in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Other techniques
for generating an immunoglobulin with a variant hinge region include
synthesizing an
oligonucleotide that encodes a hinge region, where the codon for the cysteine
to be
substituted is replaced with a codon for the substitute amino acid. This
oligonucleotide can
then be ligated into a vector backbone comprising other appropriate antibody
sequences,
such as variable regions and Fc sequences, as appropriate.
[0183] In another embodiment, a hinge cysteine can be deleted. Amino acid
deletion can
be achieved by standard molecular biology techniques, such as site directed
mutagenesis of
the nucleic acid sequence encoding the hinge region that is to be modified.
Suitable
techniques include those described in Sambrook et al., supra. Other techniques
for
generating an immunoglobulin with a variant hinge region include synthesizing
an
oligonucleotide comprising a sequence that encodes a hinge region in which the
codon for the
cysteine to be modified is deleted. This oligonucleotide can then be ligated
into a vector
backbone comprising other appropriate antibody sequences, such as variable
regions and Fc
sequences, as appropriate.
F. Bispecific Antibodies Formed Using "Protuberance-Into-Cavity" Strategy
[0184] In some embodiments, bispecific antibodies of the invention are formed
using a
"protuberance-into-cavity" strategy, also referred to as "knobs into holes"
that serves to
engineer an interface between a first and second polypeptide for hetero-
oligomerization. The
preferred interface comprises at least a part of the CH3 domain of an antibody
constant
domain. The "knobs into holes" mutations in the CH3 domain of an Fc sequence
has been
reported to greatly reduce the formation of homodimers (See, for example,
Merchant et al.,
1998, Nature Biotechnology, 16:677-681). "Protuberances" are constructed by
replacing small
amino acid side chains from the interface of the first polypeptide with larger
side chains (e.g.
tyrosine or tryptophan). Compensatory "cavities" of identical or similar size
to the
protuberances are optionally created on the interface of the second
polypeptide by replacing
large amino acid side chains with smaller ones (e.g. alanine or threonine).
Where a suitably
positioned and dimensioned protuberance or cavity exists at the interface of
either the first or
second polypeptide, it is only necessary to engineer a corresponding cavity or
protuberance,
respectively, at the adjacent interface. The protuberance and cavity can be
made by synthetic
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means such as altering the nucleic acid encoding the polypeptides or by
peptide synthesis.
For further description of knobs into holes, see U.S. Pat. Nos. 5,731,168;
5,807,706;
5,821,333.
[0185] In some embodiments "knobs into holes" technology is used to promote
heterodimerization to generate full-length bispecific anti-FcyRIIB and anti-
"activating receptor"
(e.g., IgER) antibody. In one embodiment, constructs were prepared for the
anti-Fcyl IB
component (e.g., p5A6.11.Knob) by introducing the "knob" mutation (T366W) into
the Fc
region, and the anti-IgER component (e.g., p22E7.11.Hole) by introducing the
"hole"
mutations (T3665, L368A, Y407V). In another embodiment, constructs are
prepared for the
anti-Fcyl IB component (e.g., p5A6.11.Hole) by introducing a "hole" mutation
into its Fc region,
and the anti-IgER component (e.g., p22E7.11.Knob) by introducing a "knob"
mutation in its Fc
region such as by the procedures disclosed herein or the procedures disclosed
by Merchant
et al., (1998), supra, or in U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333.
[0186] A general method of preparing a heteromultimer using the "protuberance-
into-
cavity" strategy comprises expressing, in one or separate host cells, a
polynucleotide
encoding a first polypeptide that has been altered from an original
polynucleotide to encode a
protuberance, and a second polynucleotide encoding a second polypeptide that
has been
altered from the original polynucleotide to encode the cavity. The
polypeptides are expressed,
either in a common host cell with recovery of the heteromultimer from the host
cell culture, or
in separate host cells, with recovery and purification, followed by formation
of the
heteromultimer. In some embodiments, the heteromultimer formed is a multimeric
antibody,
for example a bispecific antibody. See also US Patent Application Serial
Number 13/092,708
filed 22 April 2011.
[0187] In some embodiments, antibodies of the present invention combine a
knobs into
holes strategy with variant hinge region constructs to produce hingeless
bispecific antibodies.
G. lmmunoconjugates
[0188] The invention also provides immunoconjugates comprising an anti- IL-113
antibody
and/or anti-IL-18 antibody/antibodies herein conjugated to one or more
cytotoxic agents, such
as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g.,
protein toxins,
enzymatically active toxins of bacterial, fungal, plant, or animal origin, or
fragments thereof),
or radioactive isotopes.
[0189] In one embodiment, an immunoconjugate is an antibody-drug conjugate
(ADC) in
which an antibody is conjugated to one or more drugs, including but not
limited to a
maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP
0 425
235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and
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WO 2012/021773
PCT/US2011/047532
MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a
dolastatin; a
calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374,
5,714,586, 5,739,116,
5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res.
53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an
anthracycline
such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-
523 (2006);
Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et
al., Bioconj.
Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834
(2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et
al., J. Med.
Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate;
vindesine; a
taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and
001065.
[0190] In another embodiment, an
immunoconjugate comprises an antibody as described
herein conjugated to an enzymatically active toxin or fragment thereof,
including but not
limited to diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-
sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII,
and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes.
[0191] In another embodiment, an
immunoconjugate comprises an antibody as described
herein conjugated to a radioactive atom to form a radioconjugate. A variety of
radioactive
isotopes are available for the production of radioconjugates. Examples include
At211, 1131, 1125,
Y90, Re186 , Re188 , Sm13 , 212 , P32, Pb212 and radioactive isotopes of Lu.
When the 5 .
radioconjugate is used for detection, it may comprise a radioactive atom for
scintigraphic
studies, for example tc99m or 1123, or a spin label for nuclear magnetic
resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as iodine-123
again, iodine-
131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or
iron.
[0192] Conjugates of an antibody and
cytotoxic agent may be made using a variety of
bifunctional protein coupling agents such as N-succinimidy1-3-(2-
pyridyldithio) propionate
(SPDP), succinimidy1-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HO!),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), bis-azido
compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such
as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene
2,6-
diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene).
For example, a ricin immunotoxin can be prepared as described in Vitetta et
al., Science
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WO 2012/021773 CA 02808185 2013-02-12PCT/US2011/047532
238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzy1-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for
conjugation of
radionucleotide to the antibody. See W094/11026. The linker may be a
"cleavable linker"
facilitating release of a cytotoxic drug in the cell. For example, an acid-
labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-
containing linker
(Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may
be used.
[0193] The immunuoconjugates or ADCs herein expressly contemplate, but are not
limited to such conjugates prepared with cross-linker reagents including, but
not limited to,
BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,
and
sulfo-SMPB, and SVSB (succinimidy1-(4-vinylsulfone)benzoate) which are
commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).
II. Vectors, Host Cells and Recombinant Methods
[0194] The invention also provides isolated polynucleotides encoding the
antibodies as
disclosed herein, vectors and host cells comprising the polynucleotides, and
recombinant
techniques for the production of the antibodies.
[0195] For recombinant production of the antibody, a polynucleotide encoding
the
antibody is isolated and inserted into a replicable vector for further cloning
(amplification of
the DNA) or for expression. DNA encoding the antibody is readily isolated and
sequenced
using conventional procedures, for example, by using oligonucleotide probes
capable of
binding specifically to genes encoding the antibody. Many vectors are
available. The vector
components generally include, but are not limited to, one or more of the
following: a signal
sequence, an origin of replication, one or more marker genes, an enhancer
element, a
promoter, and a transcription termination sequence.
(i) Signal Sequence Component
[0196] The antibodies of this invention may be produced recombinantly, not
only directly,
but also as fusion antibodies with heterologous antibodies. In one embodiment,
the
heterologous antibody is a signal sequence or other antibody having a specific
cleavage site
at the N-terminus of the mature protein or antibody. The heterologous signal
sequence
selected preferably is one that is recognized and processed (i.e., cleaved by
a signal
peptidase) by the host cell. For prokaryotic host cells that do not recognize
and process the
native antibody signal sequence, the signal sequence is substituted by a
prokaryotic signal
sequence selected, for example, from the group of the alkaline phosphatase,
penicillinase,
1pp, or heat-stable enterotoxin ll leaders. For yeast secretion the native
signal sequence may
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be substituted by, e.g., the yeast invertase leader, a factor leader
(including Saccharomyces
and Kluyveromyces a-factor leaders), or acid phosphatase leader, the C.
albicans
glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell
expression,
mammalian signal sequences as well as viral secretory leaders, for example,
the herpes
simplex gD signal, are available. The DNA for such precursor region is ligated
in reading
frame to DNA encoding the antibody.
[0197] In another embodiment, production of antibodies can occur in the
cytoplasm of the
host cell, and therefore does not require the presence of secretion signal
sequences within
each cistron. In that regard, immunoglobulin light and heavy chains are
expressed, folded,
and assembled to form functional immunoglobulins within the cytoplasm. Certain
host strains
(for example, the E. coli trx13 strains) provide cytoplasm conditions that are
favorable for
disulfide bond formation, thereby permitting proper folding and assembly of
expressed protein
subunits (Proba and Plukthun, 1995, Gene, 159:203).
(ii) Origin of Replication Component
[0198] Both expression and cloning vectors contain a nucleic acid sequence
that enables
the vector to replicate in one or more selected host cells. Generally, in
cloning vectors this
sequence is one that enables the vector to replicate independently of the host
chromosomal
DNA, and includes origins of replication or autonomously replicating
sequences. Such
sequences are well known for a variety of bacteria, yeast, and viruses. The
origin of
replication from the plasmid pBR322 is suitable for most Gram-negative
bacteria, the 2
pplasmid origin is suitable for yeast, and various viral origins (5V40,
polyoma, adenovirus,
VSV, or BPV) are useful for cloning vectors in mammalian cells. Generally, the
origin of
replication component is not needed for mammalian expression vectors (the SV40
origin may
typically be used only because it contains the early promoter).
(iii) Selection Gene Component
[0199] Expression and cloning vectors may contain a selection gene, also
termed a
selectable marker. Typical selection genes encode proteins that (a) confer
resistance to
antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical nutrients not
available from
complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
[0200] One example of a selection scheme utilizes a drug to arrest growth of a
host cell.
Those cells that are successfully transformed with a heterologous gene produce
a protein
conferring drug resistance and thus survive the selection regimen. Examples of
such
dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
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[0201] Another example of suitable selectable markers for mammalian cells are
those
that enable the identification of cells competent to take up the antibody
nucleic acid, such as
DHFR, thymidine kinase, metallothionein-I and -II, preferably primate
metallothionein genes,
adenosine deaminase, ornithine decarboxylase, and the like.
[0202] For example, cells transformed with the DHFR selection gene are first
identified
by culturing all of the transformants in a culture medium that contains
methotrexate (Mtx), a
competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR
is employed
is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
[0203] Alternatively, host cells (particularly wild-type hosts that contain
endogenous
DHFR) transformed or co-transformed with DNA sequences encoding antibody, wild-
type
DHFR protein, and another selectable marker such as aminoglycoside 3'-
phosphotransferase
(APH) can be selected by cell growth in medium containing a selection agent
for the
selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin,
neomycin, or G418.
See U.S. Pat. No. 4,965,199.
[0204] A suitable selection gene for use in yeast is the trp 1 gene present in
the yeast
plasmid YRp7 (Stinchcomb et al., 1979, Nature, 282:39). The trp1 gene provides
a selection
marker for a mutant strain of yeast lacking the ability to grow in tryptophan,
for example,
ATCC No. 44076 or PEP4-1. Jones, 1977, Genetics, 85:12. The presence of the
trp1 lesion in
the yeast host cell genome then provides an effective environment for
detecting
transformation by growth in the absence of tryptophan. Similarly, Leu2-
deficient yeast strains
(for example, strains having ATCC accession number 20,622 or 38,626) are
complemented
by known plasmids bearing the Leu2 gene.
[0205] In addition, vectors derived from the 1.6 pm circular plasmid pKD1 can
be used
for transformation of Kluyveromyces yeasts. Alternatively, an expression
system for large-
scale production of recombinant calf chymosin was reported for K. lactis. See
Van den Berg,
1990, Bio/Technology, 8:135. Stable multi-copy expression vectors for
secretion of mature
recombinant human serum albumin by industrial strains of Kluyveromyces have
also been
disclosed. See Fleer et al., 1991, Bio/Technology, 9:968-975.
(iv) Promoter Component
[0206] Expression and cloning vectors usually contain a promoter that is
recognized by
the host organism and is operably linked to the antibody nucleic acid.
Promoters suitable for
use with prokaryotic hosts include the phoA promoter, 8-lactamase and lactose
promoter
systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid
promoters
such as the tac promoter. However, other known bacterial promoters are
suitable. Promoters
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for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably
linked to the DNA encoding the antibody.
[0207] Promoter sequences are known for eukaryotes. Virtually all eukaryotic
genes
have an AT-rich region located approximately 25 to 30 bases upstream from the
site where
transcription is initiated. Another sequence found 70 to 80 bases upstream
from the start of
transcription of many genes is a CNCAAT region where N may be any nucleotide.
At the 3'
end of most eukaryotic genes is an AATAAA sequence that may be the signal for
addition of
the poly A tail to the 3' end of the coding sequence. All of these sequences
are suitably
inserted into eukaryotic expression vectors.
[0208] Examples of suitable promoting sequences for use with yeast hosts
include the
promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as
enolase,
glyceraldehyde-3-phos-phate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase,
pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
[0209] Other yeast promoters, which are inducible promoters having the
additional
advantage of transcription controlled by growth conditions, are the promoter
regions for
alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative
enzymes
associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-
phosphate
dehydrogenase, and enzymes responsible for maltose and galactose utilization.
Suitable
vectors and promoters for use in yeast expression are further described in EP
73,657. Yeast
enhancers also are advantageously used with yeast promoters.
[0210] Antibody transcription from vectors in mammalian host cells is
controlled, for
example, by promoters obtained from the genomes of viruses such as polyoma
virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian
Virus 40 (SV40),
from heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin
promoter, from heat-shock promoters, provided such promoters are compatible
with the host
cell systems.
[0211] The early and late promoters of the 5V40 virus are conveniently
obtained as an
5V40 restriction fragment that also contains the 5V40 viral origin of
replication. The
immediate early promoter of the human cytomegalovirus is conveniently obtained
as a Hindi!!
E restriction fragment. A system for expressing DNA in mammalian hosts using
the bovine
papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A
modification of this
system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., 1982,
Nature 297:598-
601 on expression of human 13-interferon cDNA in mouse cells under the control
of a
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thymidine kinase promoter from herpes simplex virus. Alternatively, the rous
sarcoma virus
long terminal repeat can be used as the promoter.
(v) Enhancer Element Component
[0212] Transcription of a DNA encoding the antibody of this invention by
higher
eukaryotes is often increased by inserting an enhancer sequence into the
vector. Many
enhancer sequences are now known from mammalian genes (globin, elastase,
albumin, a-
fetoprotein, and insulin). Typically, however, one will use an enhancer from a
eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the replication
origin (bp 100-
270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the
late side of
the replication origin, and adenovirus enhancers. See also Yaniv, 1982, Nature
297:17-18 on
enhancing elements for activation of eukaryotic promoters. The enhancer may be
spliced into
the vector at a position 5' or 3' to the antibody-encoding sequence, but is
preferably located at
a site 5' from the promoter.
(vi) Transcription Termination Component
[0213] Expression vectors used in eukaryotic host cells (yeast, fungi, insect,
plant,
animal, human, or nucleated cells from other multicellular organisms) will
also contain
sequences necessary for the termination of transcription and for stabilizing
the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments
transcribed
as polyadenylated fragments in the untranslated portion of the mRNA encoding
the antibody.
One useful transcription termination component is the bovine growth hormone
polyadenylation region. See W094/11026 and the expression vector disclosed
therein.
(vii) Modulation of Translational Strength
[0214] lmmunoglobulins of the present invention can also be expressed from an
expression system in which the quantitative ratio of expressed light and heavy
chains can be
modulated in order to maximize the yield of secreted and properly assembled
full length
antibodies. Such modulation is accomplished by simultaneously modulating
translational
strengths for light and heavy chains.
[0215] One technique for modulating translational strength is disclosed in
Simmons et al.,
U.S. Pat. No. 5,840,523 and Simmons et al., 2002, J. Immunol. Methods, 263:
133-147. It
utilizes variants of the translational initiation region (TIR) within a
cistron. For a given TIR, a
series of amino acid or nucleic acid sequence variants can be created with a
range of
translational strengths, thereby providing a convenient means by which to
adjust this factor for
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the desired expression level of the specific chain. TIR variants can be
generated by
conventional mutagenesis techniques that result in codon changes which can
alter the amino
acid sequence, although silent changes in the nucleotide sequence are
preferred. Alterations
in the TIR can include, for example, alterations in the number or spacing of
Shine-Dalgamo
sequences, along with alterations in the signal sequence. One preferred method
for
generating mutant signal sequences is the generation of a "codon bank" at the
beginning of a
coding sequence that does not change the amino acid sequence of the signal
sequence (i.e.,
the changes are silent). This can be accomplished by changing the third
nucleotide position of
each codon; additionally, some amino acids, such as leucine, serine, and
arginine, have
multiple first and second positions that can add complexity in making the
bank. This method
of mutagenesis is described in detail in Yansura et al, 1992, METHODS: A
Companion to
Methods in Enzymol., 4:151-158.
[0216] Preferably, a set of vectors is generated with a range of TIR strengths
for each
cistron therein. This limited set provides a comparison of expression levels
of each chain as
well as the yield of full length products under various TIR strength
combinations. TIR
strengths can be determined by quantifying the expression level of a reporter
gene as
described in detail in Simmons et al., U.S. Pat. No. 5,840,523 and Simmons et
al., 2002, J.
Immunol. Methods, 263: 133-147. For the purpose of this invention, the
translational strength
combination for a particular pair of TIRs within a vector is represented by (N-
light, M-heavy),
wherein N is the relative TIR strength of light chain and M is the relative
TIR strength of heavy
chain. For example, (3-light, 7-heavy) means the vector provides a relative
TIR strength of
about 3 for light chain expression and a relative TIR strength of about 7 for
heavy chain
expression. Based on the translational strength comparison, the desired
individual TIRs are
selected to be combined in the expression vector constructs of the invention.
(viii) Selection and Transformation of Host Cells
[0217] Suitable host cells for cloning or expressing the DNA in the vectors
herein are the
prokaryote, yeast, or higher eukaryote cells described above. Suitable
prokaryotes for this
purpose include Archaebacteria and Eubacteria, such as Gram-negative or Gram-
positive
organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter,
Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,
Serratia, e.g., Serratia
marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B.
licheniformis 41P disclosed in DD 266,710, published 12 Apr. 1989),
Pseudomonas such as
P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli
294 (ATCC
31,446), although other strains such as E. coli B, E. coli X1776 (ATCC
31,537), and E. coli
W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than
limiting. It is
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also preferably for the host cell to secrete minimal amounts of proteolytic
enzymes, and
additional protease inhibitors may desirably be incorporated in the cell
culture. Prokaryotic
host cells may also comprise mutation(s) in the thioredoxin and/or glutathione
pathways.
[0218] In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast
are suitable cloning or expression hosts for antibody-encoding vectors.
Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used among lower
eukaryotic
host microorganisms. However, a number of other genera, species, and strains
are
commonly available and useful herein, such as Schizosaccharomyces pombe;
Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC
16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.
drosophilarum (ATCC
36,906), K. therm otolerans, and K. marxianus; yarrowia (EP 402,226); Pichia
pastoris (EP
183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;
Schwanniomyces
such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora,
Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A.
niger.
[0219] Suitable host cells for the expression of glycosylated antibody are
derived from
multicellular organisms. Examples of invertebrate cells include plant and
insect cells.
Numerous baculoviral strains and variants and corresponding permissive insect
host cells
from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
have been
identified. A variety of viral strains for transfection are publicly
available, e.g., the L-1 variant
of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such
viruses
may be used as the virus herein according to the present invention,
particularly for
transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton,
corn, potato,
soybean, petunia, tomato, and tobacco can also be utilized as hosts.
[0220] Vertebrate host cells are widely used, and propagation of vertebrate
cells in
culture (tissue culture) has become a routine procedure. Examples of useful
mammalian host
cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL
1651);
human embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK,
ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad.
Sci. USA
77:4216); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251);
monkey kidney
cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-
1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,
ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,
ATCC
CCL51); TRI cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC
5 cells; F54
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cells; mouse myeloma cells, such as NSO (e.g. RCB0213, 1992, Bio/Technology
10:169) and
SP2/0 cells (e.g. SP2/0-Ag14 cells, ATCC CRL 1581); rat myeloma cells, such as
YB2/0 cells
(e.g. YB2/3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); and a human hepatoma line
(Hep
G2). CHO cells are a preferred cell line for practicing the invention, with
CHO-K1, DUK-B11,
CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)), and
Lec13
being exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44 or CHO-
DP12 host
cells, these may be altered such that they are deficient in their ability to
fucosylate proteins
expressed therein.
[0221] The invention is also applicable to hybridoma cells. The term
"hybridoma" refers to
a hybrid cell line produced by the fusion of an immortal cell line of
immunologic origin and an
antibody producing cell. The term encompasses progeny of heterohybrid myeloma
fusions,
which are the result of a fusion with human cells and a murine myeloma cell
line subsequently
fused with a plasma cell, commonly known as a trioma cell line. Furthermore,
the term is
meant to include any immortalized hybrid cell line that produces antibodies
such as, for
example, quadromas (See, for example, Milstein et al., 1983, Nature,
537:3053). The hybrid
cell lines can be of any species, including human and mouse.
[0222] In a most preferred embodiment the mammalian cell is a non-hybridoma
mammalian cell, which has been transformed with exogenous isolated nucleic
acid encoding
the antibody of interest. By "exogenous nucleic acid" or "heterologous nucleic
acid" is meant a
nucleic acid sequence that is foreign to the cell, or homologous to the cell
but in a position
within the host cell nucleic acid in which the nucleic acid is ordinarily not
found.
(ix) Culturing the Host Cells
[0223] Host cells are transformed with the above-described expression or
cloning vectors
for antibody production and cultured in conventional nutrient media modified
as appropriate
for inducing promoters, selecting transformants, or amplifying the genes
encoding the desired
sequences.
[0224] The host cells used to produce the antibody of this invention may be
cultured in a
variety of media. Commercially available media such as Ham's F10 (Sigma),
Minimal
Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma)), and Dulbecco's Modified
Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition,
any of the
media described in Ham et al., 1979, Meth. Enz. 58:44, Barnes et al., 1980,
Anal. Biochem.
102:255, U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or
5,122,469; WO
90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media
for the
host cells. Any of these media may be supplemented as necessary with hormones
and/or
other growth factors (such as insulin, transferrin, or epidermal growth
factor), salts (such as
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sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides
(such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug),
trace
elements (defined as inorganic compounds usually present at final
concentrations in the
micromolar range), and glucose or an equivalent energy source. Any other
necessary
supplements may also be included at appropriate concentrations that would be
known to
those skilled in the art. The culture conditions, such as temperature, pH, and
the like, are
those previously used with the host cell selected for expression, and will be
apparent to the
ordinarily skilled artisan.
[0225] All culture medium typically provides at least one component from one
or more of
the following categories:
1) an energy source, usually in the form of a carbohydrate such as glucose;
2) all essential amino acids, and usually the basic set of twenty amino acids
plus
cystine;
3) vitamins and/or other organic compounds required at low concentrations;
4) free fatty acids; and
5) trace elements, where trace elements are defined as inorganic compounds or
naturally occurring elements that are typically required at very low
concentrations, usually in
the micromolar range.
[0226] The culture medium is preferably free of serum, e.g. less than about
5%,
preferably less than 1%, more preferably 0 to 0.1% serum, and other animal-
derived proteins.
However, they can be used if desired. In a preferred embodiment of the
invention the cell
culture medium comprises excess amino acids. The amino acids that are provided
in excess
may, for example, be selected from Asn, Asp, Gly, Ile, Leu, Lys, Met, Ser,
Thr, Trp, Tyr, and
Val. Preferably, Asn, Asp, Lys, Met, Ser, and Trp are provided in excess. For
example, amino
acids, vitamins, trace elements and other media components at one or two times
the ranges
specified in European Patent EP 307,247 or U.S. Pat. No. 6,180,401 may be
used. These two
documents are incorporated by reference herein.
[0227] For the culture of the mammalian cells expressing the desired protein
and capable
of adding the desired carbohydrates at specific positions, numerous culture
conditions can be
used paying particular attention to the host cell being cultured. Suitable
culture conditions for
mammalian cells are well known in the art (W. Louis Cleveland et al., 1983, J.
Immunol.
Methods 56:221-234) or can be easily determined by the skilled artisan (see,
for example,
Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B.
D., eds.
Oxford University Press, New York (1992)), and vary according to the
particular host cell
selected.
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(x) Antibody Purification
[0228] When using recombinant techniques, the antibody can be produced
intracellularly,
in the periplasmic space, or directly secreted into the medium. If the
antibody is produced
intracellularly, as a first step, the particulate debris, either host cells or
lysed fragments, is
removed, for example, by centrifugation or ultrafiltration. Carter et al.,
1992, Bio/Technology
10: 163-167 describe a procedure for isolating antibodies which are secreted
to the
periplasmic space of E. co/i. Briefly, cell paste is thawed in the presence of
sodium acetate
(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can
be removed by centrifugation. Where the antibody is secreted into the medium,
supernatants
from such expression systems are generally first concentrated using a
commercially available
protein concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A
protease inhibitor such as PMSF may be included in any of the foregoing steps
to inhibit
proteolysis and antibiotics may be included to prevent the growth of
adventitious
contaminants.
[0229] The antibody composition prepared from the cells can be purified using,
for
example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and
affinity
chromatography, with affinity chromatography being the preferred purification
technique. The
suitability of protein A as an affinity ligand depends on the species and
isotype of any
immunoglobulin Fc region that is present in the antibody. Protein A can be
used to purify
antibodies that are based on human y1, y2, or y4 heavy chains (Lindmark et
al., 1983, J.
Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and
for human y3
(Guss et al., 1986, EMBO J. 5:15671575). The matrix to which the affinity
ligand is attached is
most often agarose, but other matrices are available. Mechanically stable
matrices such as
controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow
rates and shorter
processing times than can be achieved with agarose. Where the antibody
comprises a CH3
domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful
for purification.
Other techniques for protein purification such as fractionation on an ion-
exchange column,
ethanol precipitation, Reverse Phase HPLC, chromatography on silica,
chromatography on
heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such
as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate
precipitation are also available depending on the antibody to be recovered.
[0230] In one embodiment, the glycoprotein may be purified using adsorption
onto a
lectin substrate (e.g. a lectin affinity column) to remove fucose-containing
glycoprotein from
the preparation and thereby enrich for fucose-free glycoprotein.
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(xi) Antibody Activity Assays
[0231] The immunoglobulins of the present invention can be characterized for
their
physical/chemical properties and biological functions by various assays known
in the art. In
one aspect of the invention, it is important to compare the selectivity of an
antibody of the
present invention to bind the immunogen versus other binding targets.
[0232] In certain embodiments of the invention, the immunoglobulins produced
herein
are analyzed for their biological activity. In some embodiments, the
immunoglobulins of the
present invention are tested for their antigen binding activity. The antigen
binding assays that
are known in the art and can be used herein include without limitation any
direct or
competitive binding assays using techniques such as western blots,
radioimmunoassays,
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, and protein A
immunoassays.
Illustrative antigen binding assays are provided below in the Examples
section.
[0233] The purified immunoglobulins can be further characterized by a series
of assays
including, but not limited to, N-terminal sequencing, amino acid analysis, non-
denaturing size
exclusion high pressure liquid chromatography (H PLC), mass spectrometry, ion
exchange
chromatography, and papain digestion. Methods for protein quantification are
well known in
the art. For example, samples of the expressed proteins can be compared for
their
quantitative intensities on a Coomassie-stained SDS-PAGE. Alternatively, the
specific band(s)
of interest (e.g., the full length band) can be detected by, for example,
western blot gel
analysis and/or AME5-RP assay.
III. Pharmaceutical Formulations
[0234] Therapeutic formulations of the antibody/antibodies can be prepared by
mixing
the antibody having the desired degree of purity with optional physiologically
acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A.
Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the dosages and
concentrations
employed, and include buffers such as phosphate, citrate, and other organic
acids;
antioxidants including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium
chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low
molecular weight (less than about 10 residues) antibody; proteins, such as
serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids
such as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides,
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disaccharides, and other carbohydrates including glucose, mannose, or
dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming
counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes);
and/or non-ionic
surfactants such as TWEEN TM, PLURONICSTM or polyethylene glycol (PEG).
Exemplary
pharmaceutically acceptable carriers herein further include insterstitial drug
dispersion agents
such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for
example, human
soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX , Baxter
International, Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20,
are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In
one
aspect, a sHASEGP is combined with one or more additional
glycosaminoglycanases such as
chondroitinases.
[0235] Exemplary lyophilized antibody formulations are described in US Patent
No.
6,267,958. Aqueous antibody formulations include those described in US Patent
No.
6,171,586 and W02006/044908, the latter formulations including a histidine-
acetate buffer.
[0236] The formulation herein may also contain more than one active compound
as
necessary for the particular indication being treated, preferably those with
complementary
activities that do not adversely affect each other. For instance, the
formulation may further
comprise another antibody or a chemotherapeutic agent. Such molecules are
suitably present
in combination in amounts that are effective for the purpose intended.
[0237] The active ingredients may also be entrapped in microcapsule prepared,
for
example, by coacervation techniques or by interfacial polymerization, for
example,
hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate)
microcapsule,
respectively, in colloidal drug delivery systems (for example, liposomes,
albumin
microspheres, microemulsions, nano-particles and nanocapsules) or in
macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th
edition, Osol, A.
Ed. (1980).
[0238] The formulations to be used for in vivo administration must be sterile.
This is
readily accomplished by filtration through sterile filtration membranes.
[0239] Sustained-release preparations may be prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of solid
hydrophobic
polymers containing the antibody, which matrices are in the form of shaped
articles, e.g.,
films, or microcapsule. Examples of sustained-releabe matrices include
polyesters, hydrogels
(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat.
No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-
degradable
ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such
as the LUPRON
DEPOTTm (injectable microspheres composed of lactic acid-glycolic acid
copolymer and
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leuprolide acetate), and poly-D-(¨)-3-hydroxybutyric acid. While polymers such
as ethylene-
vinyl acetate and lactic acid-glycolic acid enable release of molecules for
over 100 days,
certain hydrogels release proteins for shorter time periods. When encapsulated
antibodies
remain in the body for a long time, they may denature or aggregate as a result
of exposure to
moisture at 37 C., resulting in a loss of biological activity and possible
changes in
immunogenicity. Rational strategies can be devised for stabilization depending
on the
mechanism involved. For example, if the aggregation mechanism is discovered to
be
intermolecular S¨S bond formation through thio-disulfide interchange,
stabilization may be
achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling
moisture content, using appropriate additives, and developing specific polymer
matrix
compositions.
IV. Non-Therapeutic Uses for the Antibody
[0240] The antibody of the invention may be used as an affinity
purification agent. In this
process, the antibody is immobilized on a solid phase such a SephadexTM resin
or filter paper,
using methods well known in the art. The immobilized antibody is contacted
with a sample
containing the antigen to be purified, and thereafter the support is washed
with a suitable
solvent that will remove substantially all the material in the sample except
the antigen to be
purified, which is bound to the immobilized antibody. Finally, the support is
washed with
another suitable solvent, such as glycine buffer, pH 5.0, that will release
the antigen from the
antibody.
[0241] The antibody may also be useful in diagnostic assays, e.g., for
detecting
expression of an antigen of interest in specific cells, tissues, or serum. For
diagnostic
applications, the antibody typically will be labeled with a detectable moiety.
Numerous labels
are available which can be generally grouped into the following categories:
(a) Radioisotopes, such as 355 , 140, 1251, 3H, and 1311. 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-lnterscience, New York, N.Y., Pubs. (1991),
for example,
and radioactivity can be measured using scintillation counting.
(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.
(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
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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. Techniques for quantifying a change in
fluorescence are
described above. The chemiluminescent substrate becomes electronically excited
by a
chemical reaction and may then emit light that can be measured (using a
chemiluminometer,
for example) or donates energy to a fluorescent acceptor. 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, B-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 Enzym. (ed J. Langone
and H.
Van Vunakis), Academic press, New York, 73:147-166 (1981).
[0242] Examples of enzyme-substrate combinations include, for example:
1) Horseradish peroxidase (HRPO) utilizes hydrogen peroxide to oxidize a dye
precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl
benzidine
hydrochloride (TMB));
2) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic
substrate; and
3) B-D-galactosidase (B-D-Gal) with a chromogenic substrate (e.g., p-
nitrophenyl-B-D-
galactosidase) or fluorogenic substrate 4-methylumbelliferyl-13-D-
galactosidase.
[0243] Numerous other enzyme-substrate combinations are available to those
skilled in
the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and
4,318,980.
[0244] Sometimes, the label is indirectly conjugated with the antibody. The
skilled artisan
will be aware of various techniques for achieving this. For example, the
antibody can be
conjugated with biotin and any of the three broad categories of labels
mentioned above can
be conjugated with avidin, or vice versa. Biotin binds selectively to avidin
and thus, the label
can be conjugated with the antibody in this indirect manner. Alternatively, to
achieve indirect
conjugation of the label with the antibody, the antibody is conjugated with a
small hapten
(e.g., digoxin) and one of the different types of labels mentioned above is
conjugated with an
anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation
of the label with
the antibody can be achieved.
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[0245] In another embodiment of the invention, the antibody need not
be labeled, and the
presence thereof can be detected using a labeled antibody which binds to the
antibody.
[0246] The antibody of the present invention may be employed in any
known assay
method, such as competitive binding assays, direct and indirect sandwich
assays, and
immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of
Techniques, pp. 47-
158 (CRC Press, Inc. 1987).
[0247] The antibody may also be used for in vivo diagnostic assays.
Generally, the
antibody is labeled with a radionuclide (such as 1111n, 99TC, 140,
, .-, or 35S) so that
the antigen or cells expressing it can be localized using immunoscintiography.
V. In Vivo Uses for the Antibody
[0248] In another embodiment, the anti-IL-113 and/or anti-IL-18
antibody/antibodies of the
present invention is co-administered with a therapeutic agent to enhance the
function of the
therapeutic agent. For example, anti-FcyRIIB is administered to a mammal to
block IgG
binding to FcyRIIB, thereby preventing FcyRIIB-mediated inhibition of an
immune response.
This results in enhanced cytoxicity of an IgG therapeutic antibody. For
example, where a
therapeutic antibody is specific for a tumor antigen, co-administration of
anti-FcyRIIB of the
invention with the anti-tumor antigen antibody enhances cytoxicity of the anti-
tumor antigen
antibody.
[0249] Therapeutic antibodies, a number of which are described
above, have been
developed and approved for treatment of a variety of diseases, including
cancer. For
example, RITUXANO (Rituximab) (IDEC Pharm/Genentech, Inc.) is used to treat B
cell
lymphomas, AVASTIN TM (bevacizumab) (Genentech, Inc.) is used to treat
metastatic
colorectal cancer and HERCEPTINO (Trastumab) (Genentech, Inc.) is a humanized
anti-
HER2 monoclonal antibody used to treat metastatic breast cancer. Although, the
mechanisms
for treatment of cancer by all monoclonal antibodies developed for such
treatment may not be
completely understood, at least in some cases, a portion of the effectiveness
of antibody
therapy can be attributed to the recruitment of immune effector function
(Houghton et al.,
2000, Nature Medicine, 6:373-374; Clynes et al., 2000, Nature Medicine, 6:433-
446).
XOLAIRO (Omalizumab) (Genentech, Inc.) is an anti-IgE antibody used to treat
allergies.
[0250] The therapeutic potential for such a bifunctional antibody
would include
attenuation of signals involved in inflammation and/or allergy. For example,
when activated
by IgE and allergen (via the FcFR), mast cells and basophils secrete
inflammatory mediators
and cytokines that act on vascular and muscular cells and recruit inflammatory
cells. The
inflammatory cells in turn secrete inflammatory mediators and recruit
inflammatory cells, in a
continuing process resulting in long-lasting inflammation. Consequently, means
of controlling
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IgE induced mast cell activation provides a therapeutic approach to treating
allergic diseases
by interrupting the initiation of the inflammatory response. As described
above, a bifunctional
antibody comprises an antibody, or fragment thereof that selectively binds IL-
113 and
comprising an antibody, or fragment thereof, that selectively binds IL-18.
[0251] Additional bifunctional antibody examples (e.g, bispecific antibodies)
comprise
combinations of an antibody or fragment thereof that selectively binds IL-113,
and a second
antibody or fragment thereof, that selectively binds IL-18. In some
embodiments, the
antibody of the present invention is used to activate inhibitory FcyRIIB
receptors in a mammal
treated with the antibody so as to inhibit pro-inflammatory signals and/or B
cell activation
mediated by activating receptors. Hence, the antibody is used to treat
inflammatory disorders
and/or autoimmune diseases such as those identified above.
[0252] For the prevention or treatment of disease, the appropriate dosage of
antibody will
depend on the type of disease to be treated, the severity and course of the
disease, whether
the antibody is administered for preventive or therapeutic purposes, previous
therapy, the
patient's clinical history and response to the antibody, and the discretion of
the attending
physician. The antibody is suitably administered to the patient at one time or
over a series of
treatments.
[0253] Depending on the type and severity of the disease, about 1 pg/kg to 15
mg/kg
(e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for
administration to the patient,
whether, for example, by one or more separate administrations, or by
continuous infusion. A
typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more,
depending on the
factors mentioned above. For repeated administrations over several days or
longer,
depending on the condition, the treatment is sustained until a desired
suppression of disease
symptoms occurs. However, other dosage regimens may be useful. The progress of
this
therapy is easily monitored by conventional techniques and assays.
[0254] The antibody composition should be formulated, dosed, and administered
in a
fashion consistent with good medical practice. Factors for consideration in
this context include
the particular disorder being treated, the particular mammal being treated,
the clinical
condition of the individual patient, the cause of the disorder, the site of
delivery of the agent,
the method of administration, the scheduling of administration, and other
factors known to
medical practitioners. The "therapeutically effective amount" of the antibody
to be
administered will be governed by such considerations, and is the minimum
amount necessary
to prevent, ameliorate, or treat a disease or disorder. The antibody need not
be, but is
optionally formulated with one or more agents currently used to prevent or
treat the disorder
in question. The effective amount of such other agents depends on the amount
of antibody
present in the formulation, the type of disorder or treatment, and other
factors discussed
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above. These are generally used in the same dosages and with administration
routes as used
hereinbefore or about from 1 to 99% of the heretofore employed dosages.
[0255] For example, for treating autoimmune diseases where there is the
involvement of
an inflammatory cell (e.g., leukocyte) adhesion, migration and activation,
such as rheumatoid
arthritis and lupus, the antibody herein can be co-administered with, e.g.,
anti-LFA-1 antibody
(such as an anti-CD11a or anti-CD18 antibody) or an anti-ICAM antibody such as
ICAM-1, -2,
or -3. Additional agents for treating rheumatoid arthritis in combination with
the antibody
herein include EnbrelTM, DMARDS, e.g., methotrexate, and NSAIDs (non-steroidal
anti-
inflammatory drugs). More than one of such other active agents than the
antibody herein may
also be employed. Additionally, insulin can be used for treating diabetes,
anti-IgE for asthma,
anti-CD lla for psoriasis, anti-alpha4beta7 and growth hormone (GH) for
inflammatory bowel
disease.
[0256] Furthermore, the formulation is suitably administered along with an
effective
amount of a hypoglycemic agent. For purposes herein, the term "hypoglycemic
agent" refers
to compounds that are useful for regulating glucose metabolism, preferably
oral agents. More
preferred herein for human use are insulin and the sulfonylurea class of oral
hypoglycemic
agents, which cause the secretion of insulin by the pancreas. Examples include
glyburide,
glipizide, and gliclazide. In addition, agents that enhance insulin
sensitivity or are insulin
sensitizing, such as biguanides (including metformin and phenformin) and
thiazolidenediones
such as REZULINTM (troglitazone) brand insulin-sensitizing agent, and other
compounds that
bind to the PPAR-gamma nuclear receptor, are within this definition, and also
are preferred.
[0257] The hypoglycemic agent is administered to the mammal by any suitable
technique
including parenterally, intranasally, orally, or by any other effective route.
Most preferably, the
administration is by the oral route. For example, MICRONASETM tablets
(glyburide) marketed
by Upjohn in 1.25, 2.5, and 5 mg tablet concentrations are suitable for oral
administration.
The usual maintenance dose for Type II diabetics, placed on this therapy, is
generally in the
range of from or about 1.25 to 20 mg per day, which may be given as a single
dose or divided
throughout the day as deemed appropriate. Physician's Desk Reference, 2563-
2565 (1995).
Other examples of glyburide-based tablets available for prescription include
GLYNASETM
brand drug (Upjohn) and DIABETATm brand drug (Hoechst-Roussel). GLUCOTROLTm
(Pratt)
is the trademark for a glipizide (1-cyclohexy1-3-(p-(2-(5-methylpyrazine
carboxamide)ethyl)phenyl)sulfonyl)urea) tablet available in both 5- and 10-mg
strengths and
is also prescribed to Type II diabetics who require hypoglycemic therapy
following dietary
control or in patients who have ceased to respond to other sulfonylureas.
Physician's Desk
Reference, 1902-1903 (1995). Other hypoglycemic agents than sulfonylureas,
such as the
biguanides (e.g., metformin and phenformin) or thiazolidinediones (e.g.,
troglitozone), or other
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drugs affecting insulin action may also be employed. If a thiazolidinedione is
employed with
the peptide, it is used at the same level as currently used or at somewhat
lower levels, which
can be adjusted for effects seen with the peptide alone or together with the
dione. The typical
dose of troglitazone (REZULIN Tm) employed by itself is about 100-1000 mg per
day, more
preferably 200-800 mg/day, and this range is applicable herein. See, for
example, Ghazzi et
al., Diabetes, 46: 433-439 (1997). Other thiazolidinediones that are stronger
insulin-
sensitizing agents than troglitazone would be employed in lower doses.
VI. Articles of Manufacture
[0258] In another aspect of the invention, an article of manufacture
containing materials
useful for the treatment, prevention and/or diagnosis of the disorders
described above is
provided. The article of manufacture comprises a container and a label or
package insert on
or associated with the container. Suitable containers include, for example,
bottles, vials,
syringes, IV solution bags, etc. The containers may be formed from a variety
of materials
such as glass or plastic. The container holds a composition which is by itself
or combined
with another composition effective for treating, preventing and/or diagnosing
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 an antibody of the invention. The label or package
insert indicates
that the composition is used for treating the condition of choice. Moreover,
the article of
manufacture may comprise (a) a first container with a composition contained
therein, wherein
the composition comprises an antibody of the invention; and (b) a second
container with a
composition contained therein, wherein the composition comprises a further
cytotoxic or
otherwise therapeutic agent. The article of manufacture in this embodiment of
the invention
may further comprise a package insert indicating that the compositions can be
used to treat a
particular condition. 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 (SWF!), 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.
[0259] Therapeutic antibody compositions generally are placed into a container
having a
sterile access port, for example, an intravenous solution bag or vial having a
stopper
pierceable by a hypodermic injection needle.
[0260] The invention further provides an article of manufacture and kit
containing
materials useful for the treatment of cancer or a disease, for example. The
article of
manufacture comprises a container with a label. Suitable containers include,
for example,
bottles, vials, and test tubes. The containers may be formed from a variety of
materials such
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as glass or plastic. The container holds a composition comprising the antibody
described
herein. The active agent in the composition is the particular antibody. The
label on the
container indicates that the composition is used for the treatment or
prevention of a particular
disease or disorder, and may also indicate directions for in vivo, such as
those described
above.
[0261] The kit of the invention comprises the container described above and a
second
container comprising a buffer. It may further include other materials
desirable from a
commercial and user standpoint, including other buffers, diluents, filters,
needles, syringes,
and package inserts with instructions for use.
[0262] It is understood that any of the above articles of manufacture may
include an
immunoconjugate of the invention in place of or in addition to an IL-1[3
and/or IL-18
antibody/antibodies.
EXAMPLES
[0263] The following are examples of methods and compositions of the
invention, and
are provided herein for illustrative purposes, and are not intended to limit
the scope of the
present invention. It is understood that various other embodiments may be
practiced, given
the general description provided herein. The disclosures of all patent and
scientific literatures
cited herein are expressly incorporated in their entirety by reference.
[0264] Commercially available reagents referred to in the examples were used
according
to manufacturer's instructions unless otherwise indicated. The source of those
cells identified
in the following examples, and throughout the specification, by ATCC accession
numbers is
the American Type Culture Collection, Manassas, VA.
Methods
Dextran sodium sulfate (DSS)-induced colitis
[0265] Age and sex-matched wild-type and knock-out mice between 19-25 grams in
weight will remain untreated or receive 3.5 % DSS in their drinking water ad
lib for 5 days.
The mice will be scored daily for weight loss starting on day 4 and sacrificed
on day 8. The
colons will then be collected, scored, and used for organ culture or
histopathology. Scoring of
colons for degree of inflammation was done as follows: After removal of the
feces by
extensive flushing, the colons are scored based on the extent of wall
thickening. The score
ranges from 0 to 4, with normal colons scored as 0 and colons with thickened
wall covering
the entire length as 4.
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Colon culture for cytokine profiling
[0266] Colons from mice are cleaned, opened longitudinally, and placed in RPM!
medium
containing 1% penicillin/streptomycin solution. After overnight incubation at
37 C, the culture
medium is collected and clarified prior to cytokine analysis by xMAP-based
technology
(Luminex) using BioRad Bio-Plex single or 23-plex assays.
AAV2/5 subretinal injection
[0267] Animals were anesthetized by intraperitoneal injection of
ketamine/xylazine
(80mg/kg:15 mg/kg). Under a dissecting microscope, a 30g insulin needle was
used to create
a puncture through the sclera, allowing for subretinal injection of 1 pl of 1
x 1012 AAV2/5
genomic particles/ml (Genedetect, Bradenton, FL) using a 33-gauge Hamilton
needle and a
micro-auto-injector (World Precision Instruments, Sarasota, FL). Formation of
a subretinal
bubble indicated a successful injection.
Western Blots
[0268] Ten week old Balbc mice were injected subretinally with 1 pl of 1x109
AAV2/5-
1L113 or AAV2/5 empty control virus (Genedetect). After 4 months infection,
some mice
underwent intense light exposure (ILE, 8000 lux) for 3.5 hours then placed in
the dark for 48
hours. Eyes were dissected and eye cups (eye minus cornea and lens) were
minced in cell
lysis buffer (Cell Signaling Technologies, Danvers, MA) containing protease
inhibitors
(Protease Inhibitor Cocktail set 1, Calbiochem, Gibbstown, NJ) for 1 hr and
frozen at -80 C.
BCA assay (Thermo/Pierce, Rockford, IL) quantified protein in samples. Ten ug
protein boiled
3 minutes in Lammeli's buffer plus b-mercaptoethanol was loaded per lane in 10-
20% tris-
glycine gel (lnvitrogen, Carlsbad, CA) and run at 125 mW for 1.5 hours.
Proteins transferred
to 0.2 um pore nitrocellulose membrane in transfer buffer (Invitrogen) 25 mW
for 1 hour.
Blots were blocked with 5% milk in PBS/0.1% Tween-20 (PBST) 1 hour and 0.2
ug/ml goat-
anti-IL-113 (R&D cat# AF-401-NA) or 0.5 ug/mIrat-anti-caspase-1 (Genentech,
clone 464.2)
added in 1% milk/PBST 1 hour. Blots were washed 4x5 minutes in PBST. Anti-goat-
HRP
(1:5000 R&D) or anti-rat-HRP (Thermo/Pierce) was added for 45 minutes and
washed 5x5
minutes in PBST. Blots were developed with ECL Plus and hyperfilm (GE
Healthcare,
Buckinghamshire, UK).
Immunohistochemistry
[0269] Ten week old Balbc mice were injected subretinally with 1 pl of 1x109
AAV2/5-
1L113 or AAV2/5 empty control virus (Genedetect). After 7 weeks infection,
whole eye was
removed and fixed in 10% neutral buffered formalin overnight at room
temperature. Sections
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were processed and embedded in paraffin then stained with anti-CD45 and
visualized with
DAB.
Fluorescence Angiography (FA)
[0270] Animals were anesthetized by intraperitoneal injection of
ketamine/xylazine
(80mg/kg:15 mg/kg) and eyes dilated with 1% tropicamide (Bausch and Lomb,
Rochester,
NY). Eyes were kept moist with artificial tears. Mice were injected
intraperitoneally with 100
p110% AK-Fluor injectable fluorescein (Akorn, Buffalo Grove, IL). Images were
acquired with
a Heidelberg Spectralis HRA/OCT camera (Heidelberg Engineering, Vista, CA).
Optical Coherence Tomography (OCT)
[0271] Animals were anesthetized by intraperitoneal injection of
ketamine/xylazine
(80mg/kg:15 mg/kg) and eyes dilated with 1% tropicamide (Bausch and Lomb,
Rochester,
NY). Eyes were kept moist with artificial tears. Images were acquired with a
Heidelberg
Spectralis HRA/OCT camera (Heidelberg Engineering, Vista, CA). Measurements
are an
average thickness of 19 sections over a 15.2 mm2 area of retina. Thickness
includes retinal
and choroid.
Electroretino gram (ERG) recordings
[0272] Mice were dark adapted for 24 hours before ERG to equilibrate retinal
responses.
Once dark adapted, all subsequent procedures will be performed in the dark
with only a red
light for illumination. Animals were anesthetized with intraperitoneal
injection of Ketamine and
Xylazine (75-80 mg/kg :7.5-15 mg/kg). Mouse body temperature was maintained at
37 C
using a homeothermic heating plate connected to its control unit. Pupils were
dilated with 1%
atropine and the corneal surface was anesthetized with a drop of 0.5%
proparacaine HCI.
ERGs from both eyes were recorded simultaneously using an Espion E2 (Diagnosys
LLC,
Lowell, MA) visual electrophysiology system. Mice were placed on a platform
and a reference
electrode was inserted subcutaneously in the forehead and a ground electrode
was inserted
at the base of the tail. Gonak hypermellose solution was placed on the cornea
to establish an
electrical contact between the cornea and the platinum electrode and protected
eyes from
drying during the experiment. A mouse was placed in the ColorDome full field
desktop
Ganzfeld stimulator and were stimulated with white light: 3 flash intensities
ranging 1x10-5-5
cd/m2, allowing 2 minutes between flashes in order to reestablish baseline
response. Signals
were band pass-filtered at 0.15-1000 Hz and sampled at 2 kHz.
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Methods for phage panning IL-10
[0273] Several phage display synthetic antibody libraries were panned against
immobilized human IL-113. Enrichment of antibody displaying phage pools
specific for IL-113
was determined at round three and subsequent rounds by measuring the ratio of
recovered
pools of phage clones specific for IL-1[3 over those specific for binding
bovine serum albumin.
Construction of the synthetic naïve antibody phage display libraries is
described elsewhere
(Sidhu et al., 2004). After several rounds of panning, phage clones displaying
antibody
variable heavy and light chain domains specific for IL-113 were identified.
The DNA sequences
of the variable heavy and chain were determined and reformatted into human
IgG1
expression vectors to allow transient antibody expression in mammalian cells.
Antibody from
the cell culture growth media was purified using Protein A for subsequent
testing in soluble
protein binding affinity determination assays, receptor-ligand inhibition
assays and
functional cell based assays.
Competitive inhibition of human IL-10 binding to human IL-1 RI or IL-1 RhI
[0274] NeutrAvidin (Pierce, Rockford, IL) was diluted to 2 pg/mL in phosphate
buffered
saline (PBS) and coated on ELISA plates (384-well high-bind plates, Nunc,
Neptune, New
Jersey) during an overnight incubation at 4 C. After washing three times with
wash buffer
(PBS / 0.05% Tween-20), the plates were blocked with PBS / 0.5% bovine serum
albumin
(BSA) for 1 to 2 hours. This and all subsequent incubations were performed at
room
temperature on an orbital shaker. Human IL-113 (R&D Systems, Minneapolis, MN)
biotinylated
using maleimide-PEG-biotin (Pierce) according to the manufacturer's directions
was diluted to
400 ng/ml in assay buffer (PBS / 0.5% BSA / 0.05% Tween-20). The blocked
NeutrAvidin
plates were washed, and biotinylated human IL-113 was captured onto the plates
during a 1 ¨
2 hr incubation. Human IL-1R1 and IL-1R11 (R&D Systems) were labeled with
digoxigenin
(DIG) using 3-amino-3-deoxydigoxigenin hemisuccinamide succinimidyl ester
(Invitrogen,
Eugene, OR) according to the manufacturer's directions. The ability of
antibodies to block the
binding of IL-1R1 and IL-1R11 to IL-1[3 was evaluated by diluting the
antibodies over a broad
range and mixing them with equal volumes of DIG-labeled human IL-1R1 or IL-
1R11 (final
concentrations of 1 pg/ml or 60 ng/ml, respectively). The mixtures were added
to washed
plates and allowed to incubate for 1 ¨ 2 hr. Plate-bound IL-1R1 or IL-1R11 was
then detected
using a horseradish peroxidase (HRP)-conjugated monoclonal anti-DIG antibody
(Jackson
ImmunoResearch, West Grove, PA). After a 1 hr incubation and an additional
wash step,
tetramethyl benzidine (TM B, Kirkegaard & Perry Laboratories, Inc.,
Gaithersburg, MD) was
added, and color was allowed to develop for approximately 10 min. The reaction
was stopped
by the addition of 1 M phosphoric acid. The optical density was read using a
microplate
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reader (450 nm, 650 nm reference), and antibody concentrations yielding half
maximal
inhibition of binding were determined using four-parameter fits of the curves
(Kaleidagraph,
Synergy Software, Reading, PA). See Figure 21.
Competitive inhibition of mouse IL-113 binding to mouse IL-1R1 or IL-1R11
[0275] The ability of antibodies to block binding of mouse IL-1[3 to mouse IL-
IRI and IL-
1 RII was evaluated using a similar method. Mouse 1L-18 (R&D Systems) was
biotinylated
using sulfo-NHS-LC-biotin (Pierce) according to the manufacturer's directions
and captured
onto NeutrAvidin plates at a concentration of 400 ng/ml. Antibodies were
diluted over a broad
range, mixed with an equal volumes of mouse IL-IRI- or IL-IRII-human IgG1 Fc
fusion
proteins (R&D Systems; final concentrations of 1 ug/m1 or 60 ng/ml,
respectively), and
incubated for 1 ¨ 2 hr on the prepared plates. Bound receptor was detected
using an HRP-
conjugated goat polyclonal anti-human IgG Fc antibody (Jackson
ImmunoResearch). Color
development and data analysis were performed as described above. See Figure
21.
Competitive inhibition of human IL-18 binding to human IL-18Ra
[0276] The overall assay method was essentially the same as described for
evaluating
inhibition of human IL-1[3/1L-1 R binding. ELISA plates were coated with
NeutrAvidin (Pierce),
and human IL-18 (R&D Systems) biotinylated using sulfo-NHS-LC-biotin (Pierce)
was diluted
to 400 ng/ml and captured onto the plates. Human IL-18Ra-human IgG1 Fc (R&D
Systems)
was labeled with digoxigenin (DIG) using 3-amino-3-deoxydigoxigenin
hemisuccinamide
succinimidyl ester (Invitrogen, Eugene, OR). Diluted antibodies were mixed
with equal
volumes of DIG-IL-18Ra-Fc (final concentration of 1 ug/m1). Bound receptor was
detected
using an anti-DIG antibody (Jackson ImmunoResearch). Color development and
data
analysis were performed as described above.
EXAMPLE 1: Combined IL-1[3 and IL-18 blockade in Inflammatory Bowel Disease
[0277] In clinical studies, the present inventors have found a significant
increase in IL-113
and IL-18-expressing cells in Crohn's disease, as well as significantly
increased serum IL-18
levels in Crohn's disease (See Figure 4). In preclinical mouse models of IBD,
an increase in
IL-113 and IL-18 secretion from the colon in an ex-vivo colon culture was
found (see Figure 5).
For IL-1[3, positive cells are at sites of active inflammation with few or no
positive cells in
areas without evidence of active inflammation (Figures, upper photos). For IL-
18, positive
cells are morphologically compatible with follicular dendritic cells (arrows)
and myeloid
dendritic cells in the marginal zone (arrowheads) of the lymphoid follicle
(Figure 5, lower
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photos). IL-18 positive cells are also colon epithelial cells. These results
are representative of
21 Crohn's disease patient samples evaluated.
[0278] Mouse models studied included DSS-induced colitis (in WT B6 female
mice), T-
cell adoptive transfer and piroxicam-IL-10 KO (see Figures 6, 7 and 8). The
present inventors
have demonstrated that blockade of IL-113, IL-18 or both (in the case of ASC
KO studies)
reduces inflammatory response (IL-1[3, IL-18, TNFa, IL-17, IL-6) and colon
scores in the DSS
model of colitis (see Figures 9-13).
EXAMPLE 2: Combined IL-1(3 and IL-18 blockade in Age-Related Macular
Degeneration
[0279] Previous studies reported that IL-113 is increased in vitreous fluid of
patients with
diabetic retinopathy and uveitis. However, no studies have reported on the
presence of IL-1[3
and IL-18 in wet or dry AMD. The present studies show that IL-113 levels are
increased in
vitreous of a subpopulation of AMD patients (see Figure 14). In preclinical
mouse studies, the
present inventors show that over-expression of IL-113 in the mouse eye induces
retinal
inflammation, while IL-18 over-expression does not (see Figures 15-18).
Further, the present
inventors show that both, IL-113 and IL-18, affect retinal function as
measured by ERG
recordings (see Figure 19). Based on these studies, the present inventors
conclude that
single and combined IL-113 and IL-18 blockade is expected to improve
photoreceptor function
and CNV/edema (see Figure 20).
EXAMPLE 3: Combined IL-10 and IL-18 blockade in Type 2 Diabetes Mellitus
[0280] The present inventors hypothesize that targeting IL-113 may preserve 6-
cell
functions in patients with type 2 diabetes. IL-113 reportedly decreases
insulin secretion by
pancreatic 13 cells in vitro and alters various 6-cell functions. Further,
treatment with IL-1Ra
reportedly may prevent or ameliorate animal models of diabetes, and IL-1Ra is
reportedly
decreased in 13 cells obtained from patients with type 2 diabetes. See Figure
28.
[0281] Gene polymorphisms in the IL-16/1L-18 pathway are reportedly associated
with
central obesity and metabolic syndrome (Carter et al., 2008). Further, IL-1[3
reportedly
decreases insulin secretion by pancreatic 13 cells in vitro (Lewis and
Dinarello, 2006), and
Anakinra (IL-1Ra) reportedly improves glycemia and beta cell secretory
function in patients
(Larsen et al., 2007). Additionally, increased serum levels of IL-1 and IL-18,
reportedly
decreased the ratio to IL-1Ra and IL-18BP in T2DM patients, and IL-1Ra and IL-
18BP protect
against STZ or high-fat induced hyperglycemia in preclinical models (Sandberg
et al., 1994).
[0282] Further, Larsen et al. carried out a double-blind clinical trial in
patients with type 2
diabetes by administering anakinra once daily for 13 weeks (Larsen et al.,
2007). This
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PCT/US2011/047532
treatment improved glycemia and 13-cell insulin secretory capacity as well as
reduced markers
of systemic inflammation. However, there remains a need to determine the
possible
beneficial effects of anti-IL-1 therapies possessing a more prolonged half-
life and
administered over a longer period of time on restoration of 13-cell mass and
function in
patients with type 2 diabetes.
Example 4: Anti-IL-1b and/or anti-1L18 in the piroxicam IL-10K0 IBD model
[0283] IL-10-/- mice develop spontaneous colitis. However, the incidence
and severity
are inconsistent, which make it harder to be used as model for IBD to test our
therapeutics.
By feeding the IL-10-/- mice with piroxicam, it is likely that piroxicam will
exacerbate the
chronic intestinal inflammation in these mice and may synchronize the onset of
the colitis as
indicated by Berg etal. (2002). Thus, this is a chronic inflammation model of
IBD in contrast
to the acute DSS model of IBD.
[0284] 6-wk old female IL-10K0 (Genentech) mice were divided into the
following
treatment groups:
Group Agent Dose Frequency Route
7g: anti-ragweed' 1 mg/mou4:::::::3 times a week::
anti-gp120 1 mg/mouse
2 TNFRII-Fc 300ug/mouse 3 times a week i.p. 12
anti-IL-lb 1 mg/mouse 3 times a week i.p. 12
4 anti-IL-18 1 mg/mouse 3 times a week i.p. 12
6 anti-IL-lb 1 mg/mouse
3 times a week,
[0285] Piroxicam powder was mixed with powdered rodent diet at the
concentration of
200 ppm using geometric dilution. Briefly, an equivalent amount of mouse diet
was added to
the piroxicam and then mixed thoroughly. Successive equivalent amounts of the
mouse diet
were added, mixing well after each dilution, until the entire quantity of the
mouse diet was
incorporated. After overnight fasting, mice were fed on the piroxicam
containing diet for 11
days, and regular diet was put back on Day 12. All treatments were injected at
the amount
indicated above in 400 pl PBS 3 times a week i.p. for 6 weeks. Animals were
weighed daily
and sacrificed at the end of the study for analysis. Before the start of
prioxicam treatment,
100 pl of blood was collected through tail vein by tail nick for FACS and
serum. Then, 100 pl
of blood will be collected at week 5 after the start of the experiment. In
these studies,
treatment effects on visual colon score, colon histology and serum PK were
analyzed. See
Figure 29.
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[0286] Levels of various cytokines were measured in IL-10 KO mice with and
without
piroxicam treatment. See Figure 13. As noted, there is an elevation of IL-18
and IL-18 in the
piroxicam treatment group (relative to the WT animals) while TNFa, IL-12 and
IL-17 were
comparable between the groups.
[0287] For histopathologic analyses, tissues were fixed in 10% formalin and
subsequently embedded in paraffin for sectioning and haematoxylin and eosin
staining.
Histopathology scores were assessed in the proximal, medial and distal colon
as well as the
rectum and scored on a scale of 1 to 3. The scores for the individual colon
segments were
summed to yield the total score per animal. The same individual scored all
histologic features
and had no knowledge of the experimental groups.
[0288] Results for the visual colon scores as well as the histology scores are
shown in
Figure 30. Serum levels were elevated for IL-18 and IL-18. Treatment with a
combination of
anti-IL-113 and anti-IL-18 antibodies resulted in statistically significant
reduction of injury to the
colon. The combination treatment was as effective as TNFRII-Fc treatment.
These results
demonstrate that the combined blockade of IL-1[3 and IL-18 can be an effective
therapy for
IBD. Combined blockade of IL-113 and IL-18 may also provide a safer treatment
than TNF-
alpha blockade.
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References
Arend, W.P., G. Palmer, and C. Gabay. 2008. IL-1, IL-18, and IL-33 families of
cytokines.
Immunol Rev. 223:20-38.
Baldassano, R.N., J.P. Bradfield, D.S. Monos, C.E. Kim, J. T. Glessner, T.
Casalunovo, E.C.
Frackelton, F. G. Otieno, S. Kanterakis, J.L. Shaner, R.M. Smith, A. W.
Eckert, L.J. Robinson,
C.C. Onyiah, D.J. Abrams, R.M. Chiavacci, R. Skraban, M. Devoto, S.F. Grant,
and H.
Hakonarson. 2007. Association of the T300A non-synonymous variant of the
ATG16L1 gene
with susceptibility to paediatric Crohn's disease. Gut. 56:1171-3.
Cadwell, K., J. Y. Liu, S.L. Brown, H. Miyoshi, J. Loh, J.K. Lennerz, C.
Kishi, W. Kc, J.A.
Carrero, S. Hunt, C.D. Stone, E.M. Brunt, R.J. Xavier, B.P. Sleckman, E. Li,
N. Mizushima,
T. S. Stappenbeck, and H.W.t. Virgin. 2008. A key role for autophagy and the
autophagy gene
Atg16I1 in mouse and human intestinal Paneth cells. Nature. 456:259-63.
Carter, K. W., J. Hung, B.L. Powell, S. Wiltshire, B. T. Foo, Y.C. Leow, B.M.
McQuillan, M.
Jennens, P.A. McCaskie, P.L. Thompson, J.P. Beilby, and L.J. Palmer. 2008.
Association of
Interleukin-1 gene polymorphisms with central obesity and metabolic syndrome
in a coronary
heart disease population. Hum Genet. 124:199-206.
Cassel, S.L., S. Joly, and F.S. Sutterwala. 2009. The NLRP3 inflammasome: A
sensor of
immune danger signals. Semin Immunol.
Ferrero-Miliani, L., O.H. Nielsen, P.S. Andersen, and S.E. Girardin. 2007.
Chronic
inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation.
Clin Exp
Immunol. 147:227-35.
Kowluru, R.A., and S. Odenbach. 2004. Role of interleukin-1beta in the
pathogenesis of
diabetic retinopathy. Br J Ophthalmol. 88:1343-7.
Kuballa, P., A. Huett, J.D. Rioux, M.J. Daly, and R.J. Xavier. 2008. Impaired
autophagy of an
intracellular pathogen induced by a Crohn's disease associated ATG16L1
variant. PLoS One.
3:e3391.
Larsen, C.M., M. Faulenbach, A. Vaag, A. Volund, J.A. Ehses, B. Seifert, T.
Mandrup-
Poulsen, and M. Y. Donath. 2007. Interleukin-1-receptor antagonist in type 2
diabetes mellitus.
N Engl J Med. 356:1517-26.
Lewis, E.C., and C.A. Dinarello. 2006. Responses of IL-18- and IL-18 receptor-
deficient
pancreatic islets with convergence of positive and negative signals for the IL-
18 receptor.
Proc Natl Acad Sci U SA. 103:16852-7.
Ludwiczek, 0., A. Kaser, D. Novick, C.A. Dinarello, M. Rubinstein, and H.
Tilg. 2005. Elevated
systemic levels of free interleukin-18 (IL-18) in patients with Crohn's
disease. Eur Cytokine
Netw. 16:27-33.
Ludwiczek, 0., E. Vannier, I. Borggraefe, A. Kaser, B. Siegmund, C.A.
Dinarello, and H. Tilg.
2004. Imbalance between interleukin-1 agonists and antagonists: relationship
to severity of
inflammatory bowel disease. Clin Exp Immunol. 138:323-9.
Monteleone, G., F. Trapasso, T. Parrello, L. Biancone, A. Stella, R. luliano,
F. Luzza, A.
Fusco, and F. Pallone. 1999. Bioactive IL-18 expression is up-regulated in
Crohn's disease. J
Immunol. 163:143-7.
74
CA 02808185 2013-02-12
WO 2012/021773 PCT/US2011/047532
Perrier, S., F. Darakhshan, and E. Hajduch. 2006. IL-1 receptor antagonist in
metabolic
diseases: Dr Jekyll or Mr Hyde? FEBS Lett. 580:6289-94.
Saitoh, T., N. Fujita, M.H. Jang, S. Uematsu, B. G. Yang, T. Satoh, H. Omori,
T. Noda, N.
Yamamoto, M. Komatsu, K. Tanaka, T. Kawai, T. Tsujimura, 0. Takeuchi, T.
Yoshimori, and
S. Akira. 2008. Loss of the autophagy protein Atg16L1 enhances endotoxin-
induced IL-1beta
production. Nature. 456:264-8.
Sandberg, JØ, A. Andersson, D.L. Eizirik, and S. Sandler. 1994. Interleukin-
1 receptor
antagonist prevents low dose streptozotocin induced diabetes in mice. Biochem
Biophys Res
Commun. 202:543-8.
Sidhu, S.S., B. Li, Y. Chen, F.A. Fellouse, C. Eigenbrot, and G. Fuh. 2004.
Phage-displayed
antibody libraries of synthetic heavy chain complementarity determining
regions. J Mol Biol.
338:299-310.
Ten Hove, T., A. Corbaz, H. Amitai, S. Aloni, I. Belzer, P. Graber, P.
Drillenburg, S.J. van
Deventer, Y. Chvatchko, and A.A. Te Velde. 2001. Blockade of endogenous IL-18
ameliorates TNBS-induced colitis by decreasing local TNF-alpha production in
mice.
Gastroenterology. 121:1372-9.
Villani, A.C., M. Lemire, G. Fortin, E. Louis, M.S. Silverberg, C. Collette,
N. Baba, C. Libioulle,
J. Belaiche, A. Bitton, D. Gaudet, A. Cohen, D. Lan gelier, P.R. Fortin, J.E.
Wither, M. Sarfati,
P. Rutgeerts, J.D. Rioux, S. Vermeire, T.J. Hudson, and D. Franchimont. 2009.
Common
variants in the NLRP3 region contribute to Crohn's disease susceptibility. Nat
Genet. 41:71-6.
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