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CA 02467521 2004-05-14
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ANTIBODIES THAT INNB1VIUNOSPECIFICALLY
BIND TO BLyS
INTRODUCTION
[0001] The present invention relates to antibodies and related molecules that
immunospecifically bind to BLyS. The present invention also relates to methods
and
compositions for detecting, diagnosing, or prognosing a disease or disorder
associated
with aberrant BLyS or BLyS receptor expression or inappropriate function of
BLyS or
BLyS receptor, comprising antibodies or fragments or variants thereof, or
related
molecules, that immunospecifically bind to BLyS. The present invention further
relates to
methods and compositions for preventing, treating or ameliorating a disease or
disorder
associated with aberrant BLyS or BLyS receptor expression or inappropriate
BLyS
function or BLyS receptor function, comprising administering to an animal,
preferably a
human, an effective amount of one or more antibodies or fragments or variants
thereof, or
related molecules, that immunospecifically bind to BLyS.
BACKGROUND OF THE INVENTION
[0002] B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor
("TNF") superfamily that induces both ih vivo and in vitro B cell
proliferation and
differentiation (Moore et al., Science 285: 260-263 (1999)). BLyS is
distinguishable from
other B cell growth and differentiation factors such as IL-2, IL-4, IL-5, IL-
6, IL-7, IL-13,
IL-15, CD40L, or CD27L (CD70) by its monocyte-specific gene and protein
expression,
pattern and its specific receptor distribution and biological activity on B
lymphocytes.
BLyS expression is not detected on natural killer ("NK") cells, T cells or B
cells, but is
restricted to cells of myeloid origin. BLyS expression on resting monocytes is
upregulated
by interferon-gamma (IFN-gamma). The gene encoding BLyS has been mapped to
chromosome 13q34.
[0003] BLyS is expressed as a 285 amino acid type II membrane-bound
polypeptide
and a soluble 152 amino acid polypeptide (Moore et al., 1999 supra). The
membrane-
bound form of BLyS has a predicted transmembrane spanning domain between amino
acid residues 47 and 73. The NH2-terminus of the soluble form of BLyS begins
at Alals4
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of the membrane-bound form of BLyS. Soluble recombinant BLyS has been shown to
induce ih vitro proliferation of murine splenic B cells and to bind to a cell-
surface receptor
on these cells (Moore et al., 1999 supra). Soluble BLyS administration to mice
has been
shown to result in an increase in the proportion of CD45Rd°ll,
Ly6Dbnghc (also known as
ThB) B cells and an increase in serum IgM and IgA levels (Moore et al., 1999
supra).
Thus, BLyS displays a B cell tropism in both its receptor distribution and
biological
activity.
[0004] Levels of BLyS protein have been found to be elevated in patients with
autoimmune disease, including systemic lupus erythematosus (SLE), rheumatoid
arthritis,
and Sjogren's syndrome (Zhang et al., The Journal of Immunology, (2001) 166:6-
10;
Cheema et al., Arthritis and Rheumatism (2001) 44:1313-1319; and Groom et al.,
JounZal
of Clinical lyzvestigation (2002) 109:59-68). Furthermore, administration of a
soluble
form of a BLyS receptor, TACI, has 'been shown to alleviate the autoimmune
phenotype of
NZBWFl and MRL-lprllpr mice (Gross et al., Nature, (2000) 404:995-999). Thus,
antibodies and related molecules that immunospecifically bind to BLyS may find
medical
utility in, for example, the treatment of B cell disorders associated with
autoimmunity. In
other embodiments, antibodies and related molecules that immunospecifically
bind to
BLyS may find medical utility in for example, neoplasia or immunodeficiency
syndromes.
SUMMARY OF THE INVENTION
[0005] The present invention encompasses antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a polypeptide or polypeptide fragment of BLyS. In
particular,
the invention encompasses antibodies (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof) that immunospecifically
bind to a
polypeptide or polypeptide fragment of human BLyS (SEQ ID NOS:3228 and/or
3229) or
BLyS expressed on human monocytes; murine BLyS (SEQ ID NOS:3230 and/or 3231)
or
BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as
given in SEQ
ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on
the
surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides
of SEQ
ID NOS:3236 and/or 3237, the soluble form of monkey BLyS, or BLyS expressed on
2
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monkey monocytes), preferably human BLyS. The present invention also
encompasses
methods and compositions for detecting, diagnosing, or prognosing diseases or
disorders
associated with aberrant BLyS or BLyS receptor expression or inappropriate
function of
BLyS or BLyS receptor in an animal, preferably a mammal, and most preferably a
human,
comprising, or alternatively consisting of, use of antibodies (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to BLyS. Diseases and disorders which can be detected,
diagnosed, or prognosed with the antibodies (including molecules comprising,
or
alternatively consisting of, antibody fragments or variants thereof) of the
invention
include, but are not limited to, immune disorders (e.g., lupus, rheumatoid
arthritis,
multiple sclerosis, myasthenia gravis, Hashimoto's disease, and
immunodeficiency
syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and
rheumatoid
arthritis), infectious diseases (e.g., AmS), and proliferative disorders
(e.g., leukemia,
carcinoma, and lymphoma). The present invention further encompasses methods
and
compositions for preventing, treating or ameliorating diseases or disorders
associated with
aberrant BLyS or BLyS receptor expression or inappropriate function of BLyS or
BLyS
receptor in an animal, preferably a mammal, and most preferably a human,
comprising, or
alternatively consisting of, administering to said animal an effective amount
of one or
more antibodies (including molecules comprising, or alternatively consisting
of, antibody
fragments or variants thereof) that immunospecifically bind to BLyS. Diseases
and
disorders which can be prevented, treated or ameliorated by administering an
effective
amount of an antibody of the invention include, but are not limited to, immune
disorders
(e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis,
Hashimoto's
disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma,
allergic
disorders, and rheumatoid arthritis), infectious diseases (e.g., AmS), and
proliferative
disorders (e.g., leukemia, carcinoma, and lymphoma).
[0006] Using phage display technology, the present inventors have identified
single
chain antibody molecules ("scFvs") that immunospecifically bind to BLyS,
including
scFvs that immunospecifically bind to soluble BLyS, scFvs that
immunospecifically bind
the membrane-bound form of BLyS, and scFvs that immunospecifically bind to
both the
soluble form and the membrane-bound form of BLyS. Antibodies of the present
invention
are defined as able to bind the membrane bound and/or soluble forms of BLyS
according
3
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to the assays described in Examples 1 through 19. Molecules comprising, or
alternatively
consisting of, fragments or variants of these scFvs (e.g., including VH
domains, VH
CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those
referred to in Table 1), that immunospecifically bind the soluble form of
BLyS, the
membrane-bound form of BLyS, and/or both the soluble form and membrane-bound
form
of BLyS, are also encompassed by the invention, as are nucleic acid molecules
that encode
these scFvs, and/or molecules.
[0007] In particular, the invention relates to scFvs comprising, or
alternatively
consisting of, an amino acid sequence selected from the group consisting of
SEQ ID NOS:
1- 2128, preferably SEQ ID NOS:834 - 872, 1570 - 1595, and 1886 -1908, and
most
preferably SEQ ID NOS:1 - 46, 321- 329, 1563 - 1569, and 1881- 1885, as
referred to in
Table 1 below. In specific embodiments, the present invention relates to scFvs
that
immunospecifically bind the soluble form of BLyS, said scFvs comprising, or
alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1563 -
1880,
preferably SEQ ID NOS:1570 - 1595, and most preferably SEQ ID NOS: 1563 -1569,
as
referred to in Table l, below. In other embodiments, the present invention
also relates to
scFvs that immunospecifically bind the membrane-bound form of BLyS, said scFvs
comprising, or alternatively consisting of, an amino acid sequence of SEQ ID
NOS: 1881 -
2128, preferably SEQ ID NOS:1886 - 1908, and most preferably SEQ ID NOS: 1881 -
1885, as referred to in Table 1 below. The present invention further relates
to scFvs that
immunospecifically bind both the membrane-bound form and soluble form of BLyS,
said
scFvs comprising, or alternatively consisting of, an amino acid sequence of
SEQ ID NOS:
1 - 1562, preferably SEQ ID NOS: 834 - 872, and most preferably SEQ ID NOS: 1-
46,
and 321 - 329, as referred to in Table 1 below. Molecules comprising, or
alternatively
consisting of, fragments or variants of these scFvs (e.g., including VH
domains, VH
CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those
referred to in Table 1), that immunospecifically bind the soluble form of
BLyS, the
membrane-bound form of BLyS, and/or both the soluble form and membrane-bound
form
of BLyS, are also encompassed by the invention, as are nucleic acid molecules
that encode
these scFvs, and/or molecules.
[0008] The present invention provides antibodies (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof) that
4
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immunospecifically bind to a polypeptide or polypeptide fragment of BLyS, said
antibodies comprising, or alternatively consisting of, a polypeptide having
the amino acid
sequence of any one of the variable heavy ("VH") domains referred to in Table
1, below,
or any one of the variable light ("VL") domains referred to in Table 1. In a
preferred
embodiment, antibodies of the present invention comprise, or alternatively
consist of, a
polypeptide having the amino acid sequence of a VH domain contained in SEQ ll~
NOS:l
- 46, 321- 329, 834 - 872, 1563 - 1595, or 1881 - 1908, as referred to in
Table 1 below. In
another preferred embodiment, antibodies (including molecules comprising or
alternatively consisting of, antibody fragments or variants thereof) of the
present invention
comprise, or alternatively consist of, a polypeptide having the amino acid
sequence of a
VL domain contained SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or
1881 -
1908, as referred to in Table 1 below. Molecules comprising, or alternatively
consisting
of, fragments or variants of these antibodies (e.g., including VH domains, VH
CDRs, VL
domains, or VL CDRs having an amino acid sequence of any one of those referred
to in
Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-
bound
form of BLyS, and/or both the soluble form and membrane-bound form of BLyS,
are also
encompassed by the invention, as are nucleic acid molecules that encode these
antibodies,
and/or molecules.
[0009] The present invention also provides antibodies (including molecules
comprising or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS,
said
antibodies comprising, or alternatively consisting of, a polypeptide having
the amino acid
sequence of any one of the VH domains referred to in Table 1, below, and any
one of the
VL domains referred to in Table 1. In a preferred embodiment, the antibodies
of the
invention comprise or alternatively consist of, a polypeptide having the amino
acid
sequence of a VH and VL domain contained in the same scFv referred to in Table
1. In
another preferred embodiment, antibodies of the present invention, comprise,
or
alternatively consist of, a VH domain from an scFv of SEQ ID NOS:1 - 46, 321 -
329, 834
- 872, 1563 - 1595, or 1881 -1908, as disclosed in Table 1, and a VL domain
from an
scFv SEQ ID NOS:l - 46, 321- 329, 834 - 872, 1563 - 1595, or 1881 - 1908, as
disclosed
in Table 1. In another preferred embodiment, antibodies of the present
invention
comprise, or alternatively consist of, the VH and VL domain from a single scFv
of SEQ
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ll~ NOS:1- 46, 321- 329, 834 - 872, 1563 - 1595, or 1881 -1908, as disclosed
in Table 1.
Molecules comprising, or alternatively consisting of, fragments or variants of
these
antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having
an
amino acid sequence of any one of those referred to in Table 1), that
immunospecifically
bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both
the
soluble form and membrane-bound form of BLyS, are also encompassed by the
invention,
as are nucleic acid molecules that encode these antibodies, and/or molecules.
[0010] The present invention also provides antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS,
said
antibodies comprising, or alternatively consisting of, a polypeptide having
the amino acid
sequence of any one, two, three or more of the VH complementarity determining
regions
("CDRs") (i.e., VH CDRl, VH CDR2, or VH CDR3) referred to in Table 1 and/or
any
one, two, three or more of the VL CDRs (i.e., VL CDR1, VL CDR2, or VL CDR3)
referred to in Table 1. In one embodiment, antibodies of the present invention
comprise,
or alternatively consist of, a polypeptide having the amino acid sequence of
any one of the
VH CDRls referred to in Table 1 and/or any one of the VL CDRls referred to in
Table 1.
In another embodiment, antibodies of the present invention comprise, or
alternatively
consist of, a polypeptide having the amino acid sequence of any one of the VH
CDR2s
referred to in Table 1 and/or any one of the VL CDR2s referred to in Table 1.
In a
preferred embodiment, antibodies of the present invention comprise, or
alternatively
consist of, a polypeptide having the amino acid sequence of any one of the VH
CDR3s
referred to in Table 1 and/or any one of the VL CDR3s referred to in Table 1.
Molecules
comprising, or alternatively consisting of, fragments or variants of these
antibodies (e.g.,
including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid
sequence of any one of those referred to in Table 1), that immunospecifically
bind the
soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble
form
and membrane-bound form of BLyS, are also encompassed by the invention, as are
nucleic acid molecules that encode these antibodies, and/or molecules.
[0011] In another embodiment, antibodies of the present invention (including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof) immunospecifically bind to a polypeptide or polypeptide fragment of
BLyS, and
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comprise, or alternatively consist of, a polypeptide having the amino acid
sequence of any
one of the VH CDRls referred to in Table l, any one of the VH CDR2s referred
to in
Table 1, and/or any one of the VH CDR3s referred to in Table 1. In another
embodiment,
antibodies of the present invention comprise, or alternatively consist of, a
polypeptide
having the amino acid sequence of any one of the VL CDRls referred to in Table
1, any
one of the VL CDR2s referred to in Table 1, and/or any one of the VL CDR3s
referred to
in Table 1. In a preferred embodiment, antibodies of the present invention
comprise, or
alternatively consist of, at least one, two, three, four, five, six, or more
CDRs that
correspond to the same scFv referred to in Table 1, more preferably where
CDR1, CDR2,
and CDR3 of the VL domain correspond to the same scFv or where CDR1, CDR2, and
CDR3 of the VH domain correspond to the same scFv, and most preferably where
all six
CDRs correspond to the same scFv referred to in Table 1. Molecules comprising,
or
alternatively consisting of, fragments or variants of these antibodies (e.g.,
including VH
domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any
one
of those referred to in Table 1), that immunospecifically bind the soluble
form of BLyS,
the membrane-bound form of BLyS, and/or both the soluble form and membrane-
bound
form of BLyS, are also encompassed by the invention, as are nucleic acid
molecules that
encode these antibodies, and/or molecules.
[0012] The present invention also provides antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that:
immunospecifically bind.to the soluble form of BLyS (e.g., a polypeptide
consisting of
amino acids 134 - 285 of SEQ ID N0:3228); that immunospecifically bind to the
membrane-bound form of BLyS (e.g., a polypeptide consisting of amino acids 1 -
285 of
SEQ ID N0:3228 or a BLyS polypeptide expressed on the surface of monocytes)
and/or
that immunospecifically bind to both the soluble form and membrane-bound form
of
BLyS. In a preferred embodiment, antibodies of the present invention
immunospecifically
bind to the soluble form of BLyS and comprise, or alternatively consist of, a
VH domain,
VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, and/or VL CDR3
corresponding to one or more scFvs, that immunospecifically bind to the
soluble form of
~BLyS. In another preferred embodiment, antibodies of the present invention
immunospecifically bind to the membrane-bound form of BLyS and comprise, or
alternatively consist of, a VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain,
VL
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CDR1, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that
immunospecifically bind to the membrane-bound form of BLyS. In yet another
preferred
embodiment, antibodies of the present invention immunospecifically bind to the
soluble
form and membrane-bound form of BLyS and comprise, or alternatively consist
of, a VH
domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, andlor VL
CDR3 corresponding to one or more scFvs, that immunospecifically binds to the
soluble
form and membrane-bound form of BLyS. In another preferred embodiment,
antibodies
of the present invention comprise, or alternatively consist of, a VH domain
and a VL
domain corresponding to the same scFv disclosed in Table 1, which antibodies
immunospecifically bind to the soluble form of BLyS, the membrane-bound form
of
BLyS, or both the soluble form and membrane-bound form of BLyS. Nucleic acid
molecules encoding these antibodies are also encompassed by the invention.
Molecules
comprising, or alternatively consisting of, fragments or variants of these
antibodies (e.g.,
including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid
sequence of any one of those referred to in Table 1), that immunospecifically
bind the
soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble
form
and membrane-bound form of BLyS, are also encompassed by the invention, as are
nucleic acid molecules that encode these antibodies, and/or molecules.
[0013] The present invention also provides antibodies (including molecules
comprising or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to both BLyS and APRIL (preferably to the soluble
forms of
each of these molecules), said antibodies comprising, or alternatively
consisting of, a
polypeptide having the amino acid sequence of any one of the VH domains
referred to in
Table 1, below, and any one of the VL domains referred to in Table 1. In a
preferred
embodiment, the antibodies of the invention comprise or alternatively consist
of, a
polypeptide having the amino acid sequence of a VH and VL domain contained in
the
same scFv referred to in Table 1. In another preferred embodiment, antibodies
of the
present invention that immunospecifically bind to both BLyS and APRIL,
comprise, or
alternatively consist of, a VH domain from an scFv of SEQ ID NOS:3240-3247 as
disclosed in Table 1, and a VL domain from an scFv SEQ ID NOS:3240-3247, as
disclosed in Table 1. In another preferred embodiment, antibodies of the
present invention
that immunospecifically bind to both BLyS and APRIL comprise, or alternatively
consist
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of, the VH and VL domain from a single scFv of SEQ ID NOS: SEQ )D NOS:3240-
3247,
as disclosed in Table 1. Molecules comprising, or alternatively consisting of,
fragments or
variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains,
or VL
CDRs having an amino acid sequence of any one of those referred to in Table
1), that
immunospecifically bind both BLyS and APRIL, are also encompassed by the
invention,
as are nucleic acid molecules that encode these antibodies, and/or molecules.
[0014] The present invention also provides antibodies (including molecules
comprising or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a heterotrimeric protein comprising at least one
BLyS
polypeptide (preferably amino acids 134-285 of SEQ ID NO:3228), said
antibodies
comprising, or alternatively consisting of, a polypeptide having the amino
acid sequence
of any one of the VH domains referred to in Table l, below, and any one of the
VL
domains referred to in Table 1. In a preferred embodiment, the antibodies of
the invention
that immunospecifically bind heterotrimeric protein comprising at least one
BLyS
polypeptide, comprise or alternatively consist of, a polypeptide having the
amino acid
sequence of a VH and VL domain contained in the same scFv referred to in Table
1. In
another preferred embodiment, antibodies of the present invention that
immunospecifically bind heterotrimeric protein comprising at least one BLyS
polypeptide,
comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID
NOS:3240-
3247 as disclosed in Table l, and a VL domain from an scFv SEQ ID NOS:3240-
3247, as
disclosed in Table 1. In another preferred embodiment, antibodies of the
present invention
that immunospecifically bind heterotrimeric protein comprising at least one
BLyS
polypeptide, comprise, or alternatively consist of, the VH and VL domain from
a single
scFv of SEQ ID NOS:3240-3247, as disclosed in Table 1. Molecules comprising,
or
alternatively consisting of, fragments or variants of these antibodies (e.g.,
including VH
domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any
one
of those referred to in Table 1), that immunospecifically bind a
heterotrimeric protein
comprising at least one BLyS polypeptide, are also encompassed by the
invention, as are
nucleic acid molecules that encode these antibodies, and/or molecules.
[0015] A VH domain of an amino acid sequence disclosed herein may be combined
witha VL domain of an amino acid sequence disclosed herein, or other VL
domains, to
provide a VH/VL pairing representing an antigen-binding site of an antibody.
Similarly, a
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VL domain of an amino acid sequence disclosed herein may be combined with a VH
domain of an amino acid sequence disclosed herein, or other VH domains.
Further, one or
more CDRs disclosed herein may be taken from a VH or VL domain and
incorporated into
a suitable framework as discussed infra.
[0016] The present invention provides antibodies (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof (including
derivatives))
comprising, or alternatively consisting of, of VH domains, VL domains and/or
CDRs
described herein, which antibodies, immunospecifically bind to BLyS (e.g.,
soluble BLyS
and membrane-bound BLyS) and can be routinely assayed for immunospecific
binding to
BLyS using methods known in the art, such as, for example, the immunoassays
disclosed
infra. Antibodies and antibody fragments or variants (including derivatives)
of the
invention may include, for example, one or more amino acid sequence
alterations
(addition, deletion, substitution and/or insertion of an amino acid residue).
These
alterations may be made in one or more framework regions and/or one or more
CDR's.
The antibodies of the invention (including antibody fragments, and variants
and derivative
thereof) can be routinely made by methods known in the art. Molecules
comprising, or
alternatively consisting of, fragments or variants of any of the VH domains,
VH CDRs,
VL domains, and VL CDRs whose sequences are specifically disclosed herein may
be
employed in accordance with the present invention. Nucleic acid molecules
encoding
these antibodies and molecules (including fragments, variants, and
derivatives) are also
encompassed by the invention.
[0017] The present invention also provides panels of antibodies (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants)
wherein the
panel members correspond to one, two, three, four, five, ten, fifteen, twenty,
or more
different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab')2
fragments, Fd
fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies,
and scFvs).
The present invention further provides mixtures of antibodies, wherein the
mixture
corresponds to one, two, three, four, five, ten, fifteen, twenty, or more
different antibodies
of he invention (e.g., whole antibodies, Fabs, F(ab')2 fragments, Fd
fragments, disulfide-
linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies, and scFvs)). The
present invention
also provides for compositions comprising, or alternatively consisting of,
one, two, three,
four, five, ten, fifteen, twenty, or more antibodies of the present invention
(including
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molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof). A composition of the invention may comprise, or alternatively
consist of, one,
two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of
one or more
antibodies or fragments or variants thereof. Alternatively, a composition of
the invention
may comprise, or alternatively consist of, nucleic acid molecules encoding one
or more
antibodies of the invention.
[0018] The present invention also provides for fusion proteins comprising an
antibody
(including molecules comprising, or alternatively consisting of, antibody
fragments or
variants thereof) of the invention, and a heterologous polypeptide (i.e., a
polypeptide
unrelated to an antibody or antibody domain). Nucleic acid molecules encoding
these
fusion proteins are also encompassed by the invention. A composition of the
present
invention may comprise, or alternatively consist of, one, two, three, four,
five, ten, fifteen,
twenty or more fusion proteins of the invention. Alternatively, a composition
of the
invention may comprise, or alternatively consist of, nucleic acid molecules
encoding one,
two, three, four, five, ten, fifteen, twenty or more fusion proteins of the
invention.
[0019] The present invention also provides for a nucleic acid molecule,
generally
isolated, encoding an antibody (including molecules such as scFvs, which
comprise, or
alternatively consist of, an antibody fragment or variant thereof) of the
invention. The
present invention also provides a host cell transformed with a nucleic acid
molecule of the
invention and progeny thereof. The present invention also provides a method
for the
production of an antibody (including a molecule comprising, or alternatively
consisting of,
an antibody fragment or variant thereof) of the invention. The present
invention further
provides a method of expressing an antibody (including a molecule comprising,
or
alternatively consisting of, an antibody fragment or variant thereof) of the
invention from
a nucleic acid molecule. These and other aspects of the invention are
described in further
detail below.
[0020] The present invention also encompasses methods and compositions for
detecting, diagnosing and/or prognosing diseases or disorders associated with
aberrant
BLyS or BLyS receptor expression or inappropriate BLyS or BLyS receptor
function in an
animal, preferably a mammal, and most preferably a human, comprising using
antibodies
(including molecules which comprise, or alternatively consist of, antibody
fragments or
variants thereof) that immunospecifically bind to BLyS. Diseases and disorders
which can
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be detected, diagnosed or prognosed with the antibodies of the invention
include, but are
not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple
sclerosis,
myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome),
inflammatory
disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis),
infectious diseases
(e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and
lymphoma).
[0021] In specific embodiments, the present invention encompasses methods and
compositions for detecting, diagnosing and/or prognosing diseases or disorders
associated
with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some
immunodeficiencies). In other specific embodiments, the present invention
encompasses
methods and compositions for detecting, diagnosing and/or prognosing diseases
or
disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency).
[0022] The present invention further encompasses methods and compositions for
preventing, treating or ameliorating diseases or disorders associated with
aberrant BLyS or
BLyS receptor expression or inappropriate BLyS or BLyS receptor function in an
animal,
preferably a mammal, and most preferably a human, comprising administering to
said
animal an effective amount of one or more antibodies (including molecules
which
comprise, or alternatively consist of, antibody fragments or variants thereof)
that
immunospecifically bind to BLyS. Diseases and disorders which can be
prevented, treated
or inhibited by administering an effective amount of one or more antibodies or
molecules
of the invention include, but are not limited to, immune disorders (e.g.,
lupus, rheumatoid
arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and
immunodeficiency syndrome), inflammatory disorders (e.g.,-asthma, allergic
disorders,
and rheumatoid arthritis), infectious diseases (e.g., ASS), and proliferative
disorders
(e.g., leukemia, carcinoma, and lymphoma).
[0023] In specific embodiments, the present invention encompasses methods and
compositions (e.g., antagonistic anti-BLyS antibodies) for preventing,
treating or
ameliorating diseases or disorders associated with hypergammaglobulinemia
(e.g., AIDS,
autoimmune diseases, and some immunodeficiency syndromes). In other specific
embodiments, the present invention encompasses methods and compositions (e.g.,
agonistic anti-BLyS antibodies) for preventing, treating or ameliorating
diseases or
disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency
syndrome).
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[0024] Autoimmune disorders, diseases, or conditions that may be detected,
diagnosed, prognosed, or monitored using the antibodies of the invention
include, but are
not limited to, autoimrnune hemolytic anemia, autoimrnune neonatal
thrombocytopenia,
idiopathic thrombocytopenia purpura, autoimmune neutropenia,
autoimmunocytopenia,
hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive
enteropathy,
allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic
heart disease,
glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis,
Uveitis
Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's
Disease, Stiff Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis,
IgA
glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-
Barre
Syndrome, insulin dependent diabetes rnellitis, and autoimmune inflammatory
eye,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis,
systemic lupus
erhythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor
autoimmunities such as, for example, (a) Graves' Disease , (b) Myasthenia
Gravis, and (c)
insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic
purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies,
mixed
connective tissue disease, polymyositis/dermatomyositis, pernicious anemia,
idiopathic
Addison's disease, infertility, glomerulonephritis such as primary
glomerulonephritis and
IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus,
and
adrenergic drug resistance (including adrenergic drug resistance with asthma
or cystic
fibrosis), chronic active hepatitis, primary biliary cirrhosis, other
endocrine gland failure,
vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic
dermatitis, asthma,
inflammatory myopathies, and other inflammatory, granulomatous, degenerative,
and
atrophic disorders).
[0025] Immunodeficiencies that may be detected, diagnosed, prognosed, or
monitored
using the antibodies of the invention include, but are not limited to, severe
combined
immunodeficiency (SLID)-X linked, SCID-autosomal, adenosine deaminase
deficiency
(ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease,
congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia
of
infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common
variable
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immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked
immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM,
selective IgA deficiency, IgG subclass deficiency (with or without IgA
deficiency),
antibody deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig
heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative
disorder
(BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss
type),
reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia,
thymic
alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-
telangiectasia, short
limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-
combined irnmunodeficiency with Igs, purine nucleoside phosphorylase
deficiency (PNP),
MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined
immunodeficiency.
DEFINITIONS
[0026] The term "antibody," as used herein, refers to immunoglobulin molecules
and
immunologically active portions of immunoglobulin molecules, i.e., molecules
that
contain an antigen binding site that immunospecifically binds an antigen. As
such, the
term antibody encompasses not only whole antibody molecules, but also antibody
fragments as well as variants (including derivatives) of antibodies and
antibody fragments.
Examples of molecules which are described by the term "antibody" in this
application
include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab'
fragments,
F(ab')2, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or
alternatively
consisting of, either a VL or a VH domain. The term "single chain Fv" or
"scFv" as used
herein refers to a polypeptide comprising a VL domain of antibody linked to a
VH domain
of an antibody. Antibodies that immunospecifically bind to BLyS may have cross-
reactivity with other antigens. Preferably, antibodies that immunospecifically
bind to
BLyS do not cross-react with other antigens. Antibodies that
immunospecifically bind to
BLyS can be identified, for example, by immunoassays or other techniques known
to
those of skill in the art, e.g., the immunoassays described in the Examples
below.
[0027] Antibodies of the invention include, but are not limited to,
monoclonal,
multispecific, human or chimeric antibodies, single chain antibodies, Fab
fragments,
F(ab') fragments, antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to
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antibodies of the invention), and epitope-binding fragments of any of the
above. The
immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE,
IgM, IgD,
IgA and IgY), class (e.g., IgGI, IgGz, IgG3, IgG4, IgAI and IgAz) or subclass
of
immunoglobulin molecule.
[0028] Antibodies of the invention may also include multimeric forms of
antibodies.
For example, antibodies of the invention may take the form of antibody dimers,
trimers, or
higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole
immunoglobulin molecules or of F(ab')2 fragments are tetravalent, whereas
dimers of Fab
fragments or scFv molecules are bivalent. Individual monomers withon an
antibody
multimer may be identical or different, i.e., they may be heteromeric or
homomeric
antibody rnultimers. For example, individual antibodies within a multimer may
have the
same or different binding specificities. Multimerization of antibodies may be
accomplished through natural aggregation of antibodies or through chemical or
recombinant linking techniques known in the art. For example, some percentage
of
purified antibody preparations (e.g., purified IgG1 molecules) spontaneously
form protein
aggregates containing antibody homodimers, and other higher-order antibody
multimers.
Alternatively, antibody homodimers may be formed through chemical linkage
techniques
known in the art. For example, heterobifunctional crosslinking agents
including, but not
limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate]
and
SATA [N succinimidyl S-acethylthio-acetate] (available, for example, from
Pierce
Biotechnology, Inc. (Rockford, IL)) can be used to form antibody multimers. An
exemplary protocol for the formation of antibody homodimers is given in Ghetie
et al.,
Proceedings of the National Academy of Sczences USA (1997) 94:7509-7514, which
is
hereby incorporated by reference in its entirety. Antibody homodimers can be
converted
to Fab'2 homodimers through digestion with pepsin. Another way to form
antibody
homodimers is through the use of the autophilic T15 peptide described in Zhao
and
Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby
incorporated by
reference in its entirety.
[0029] Alternatively, antibodies can be made to multimerize through
recombinant
DNA techniques. IgM and IgA naturally form antibody multimers through the
interaction
with the J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG
molecules, can
be engineered to contain the J chain interaction domain of IgA or IgM, thereby
conferring
CA 02467521 2004-05-14
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the ability to form higher order multimers on the non-IgA or non-IgM
molecules. (see, for
example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and
Frigerio et
al., (2000) Planet Physiology 123:1453-94., both of which are hereby
incorporated by
reference in their entireties.) ScFv dimers can also be formed through
recombinant
techniques known in the art; an example of the construction of scFv dimers is
given in
Goel et al., (2000) Caficer Research 60:6964-6971 which is hereby incorporated
by
reference in its entirety. Antibody multimers may be purified using any
suitable method
known in the art, including, but not limited to, size exclusion
chromatography.
[0030] Unless otherwise defined in the specification, specific binding or
immunospecifc binding by an anti-BLyS antibody means that the anti-BLyS
antibody
binds BLyS but does not significantly bind to (i.e., cross react with)
proteins other than
BLyS, such as other proteins in the same family of proteins, e.g., other TNF
family
ligands). An antibody that binds BLyS protein and does not cross-react with
other
proteins is not necessarily an antibody that does not bind said other proteins
in all
conditions; rather, the BLyS -specific antibody of the invention
preferentially binds BLyS
compared to its ability to bind said other proteins such that it will be
suitable for use in at
least one type of assay or treatment, i.e., give low background levels or
result in no
unreasonable adverse effects in treatment. It is well known that the portion
of a protein
bound by an antibody is known as the epitope. An epitope may either be linear
(i.e.,
comprised of sequential amino acids residues in a protein sequences) or
conformational
(i.e., comprised of one or more amino acid residues that are not contiguous in
the primary
structure of the protein but that are brought together by the secondary,
tertiary or
quaternary structure of a protein). Given that BLyS -specific antibodies bind
to epitopes
of BLyS, an antibody that specifically binds BLyS may or may not bind
fragments of
BLyS and/or variants of BLyS (e.g., proteins that are at least 90% identical
to BLyS)
depending on the presence or absence of the epitope bound by a given BLyS-
specific
antibody in the BLyS fragment or variant. Likewise, BLyS-specific antibodies
of the
invention may bind species orthologues of BLyS (including fragments thereof)
depending
on the presence or absence of the epitope recognized by the antibody in the
orthologue.
Additionally, BLyS-specific antibodies of the invention may bind modified
forms of
BLyS, for example, BLyS fusion proteins. In such a case when antibodies of the
invention
bind BLyS fusion proteins, the antibody must make binding contact with the
BLyS moiety
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of the fusion protein in order for the binding to be specific. Antibodies that
specifically
bind to BLyS can be identified, for example, by immunoassays or other
techniques known
to those of skill in the art, e.g., the immunoassays described in the Examples
below.
[0031] Furthermore, in the present application certain antibodies may be
specific for
either the membrane bound form of BLyS, or the soluble form of BLyS (i.e., 134-
285 of
SEQ ID N0:2, preferably trimers of proteins consisting of amino acids 134-285
of SEQ
ID N0:2), or both. Antibodies of the present invention are defined as able to
bind the
membrane bound and/or soluble forms of BLyS according to the assays described
in
Examples 1 through 19.
[0032] Preferably, an antibody of the invention comprises, or alternatively
consists of,
a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any
one of those referred to in Table l, or a fragment or variant thereof.
[0033] An antibody of the invention "which binds the soluble form of BLyS" is
one
which binds the 152 amino acid soluble form of the BLyS protein (amino acids
134- 285
of SEQ DJ N0:3228). In specific embodiments of the invention, an antibody of
the
invention "which binds the soluble form of BLyS" does not also bind the
membrane-
bound or membrane-associated form of BLyS. Assays which measure binding to the
soluble form of BLyS include, but are not limited to, receptor binding
inhibition assay or
capture of soluble BLyS from solution as described in Examples 8 and 9.
[0034] An antibody of the invention "which binds the membrane-bound form of
BLyS" is one which binds the membrane-associated (uncleaved) BLyS protein. In
specific embodiments of the invention, an antibody of the invention "which
binds the
membrane-bound form of BLyS" does not also bind the soluble form of BLyS.
Binding to
HIS-tagged BLyS (as described herein) in an ELISA is an indicator that an
antibody binds
the membrane-bound form of BLyS, but should not be relied upon as proof of
specificity
for the membrane-bound form of BLyS. Assays that may be relied upon as proof
of an
antibody's specificity for membrane-bound BLyS, include, but are not limited
to, binding
to plasma membranes expressing BLyS as described in Example 2. An antibody of
the
invention "which binds the both the soluble form and the membrane-bound form
of
BLyS" is one which binds both the membrane-bound form and the soluble form of
BLyS.
[0035] The term "variant" as used herein refers to a polypeptide that
possesses a
similar or identical function as a BLyS polypeptide, a fragment of BLyS, an
anti-BLyS
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antibody or antibody fragment thereof, but does not necessarily comprise a
similar or
identical amino acid sequence of a BLyS polypeptide, a fragment of BLyS, an
anti-BLyS
antibody or antibody fragment thereof, or possess a similar or identical
structure of a
BLyS polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody
fragment
thereof. A variant having a similar amino acid refers to a polypeptide that
satisfies at least
one of the following: (a) a polypeptide comprising, or alternatively
consisting of, an amino
acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95% or at least 99% identical to the amino acid
sequence of a
BLyS polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody
fragment
thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino
acid
sequence of any one of those referred to in Table 1) described herein; (b) a
polypeptide
encoded by a nucleotide sequence, the complementary sequence of which
hybridizes
under stringent conditions to a nucleotide sequence encoding a BLyS
polypeptide (e.g.,
SEQ >D N0:3228), a fragment of BLyS, an anti-BLyS antibody or antibody
fragment
thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino
acid
sequence of any one of those referred to in Table 1), described herein, of at
least 5 amino
acid residues, at least 10 amino acid residues, at least 15 amino acid
residues, at least 20
amino acid residues, at least 25 amino acid residues, at least 30 amino acid
residues, at
least 40 amino acid residues, at least 50 amino acid residues, at least 60
amino residues, at
least 70 amino acid residues, at least 80 amino acid residues, at least 90
amino acid
residues, at least 100 amino acid residues, at least 125 amino acid residues,
or at least 150
amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence
that is at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at
least 95% or at least 99%, identical to the nucleotide sequence encoding a
BLyS
polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody fragment
thereof
(including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid
sequence of any one of those referred to in Table 1), described herein. A
polypeptide with
similar structure to a BLyS polypeptide, a fragment of BLyS, an anti-BLyS
antibody or
antibody fragment thereof, described herein refers to a polypeptide that has a
similar
secondary, tertiary or quarternary structure of a BLyS polypeptide, a fragment
of BLyS, an
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anti-BLyS antibody, or antibody fragment thereof, described herein. The
structure of a
polypeptide can determined by methods known to those skilled in the art,
including but
not limited to, X-ray crystallography, nuclear magnetic resonance, and
crystallographic
electron microscopy.
[0036] To determine the percent identity of two amino acid sequences or of two
nucleic acid sequences, the sequences are aligned for optimal comparison
purposes (e.g.,
gaps can be introduced in the sequence of a first amino acid or nucleic acid
sequence for
optimal alignment with a second amino acid or nucleic acid sequence). The
amino acid
residues or nucleotides at corresponding amino acid positions or nucleotide
positions are
then compared. When a position in the first sequence is occupied by the same
amino acid
residue or nucleotide at the corresponding position in the second sequence,
then the
molecules are identical at that position. The percent identity between the two
sequences is
a function of the number of identical positions shared by the sequences (i.e.,
% identity =
number of identical overlapping positions/total number of positions x 100%).
In one
embodiment, the two sequences are the same length.
[0037] The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm known to those of skill in the
art. An
example of a mathematical algorithm for comparing two sequences is the
algorithm of
Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268(1990), modified as
in
Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877(1993). The BLASTn
and
BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410(1990) have
incorporated
such an alogrithm. BLAST nucleotide searches can be performed with the BLASTn
program, score = 100, wordlength = 12 to obtain nucleotide sequences
homologous to a
nucleic acid molecules of the invention. BLAST protein searches can be
performed with
the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences
homologous to a protein molecules of the invention. To obtain gapped
alignments for
comparison purposes, Gapped BLAST can be utilized as described in Altschul et
al.
Nucleic Acids Res. 25:3389-3402(1997). Alternatively, PSI-BLAST can be used to
perform an iterated search which detects distant relationships between
molecules (Id.).
When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default
parameters of the respective programs (e.g., BLASTx and BLASTn) can be used.
(See
http:/lwww.ncbi.nlm.nih.gov.)
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[0038] Another example of a mathematical algorithm utilized for the comparison
of
sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN
program
(version 2.0) which is part of the GCG sequence alignment softwaxe package has
incorporated such an alogrithm. Other algorithms for sequence analysis known
in the art
include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl.
Biosci., 10 :3-5(1994); and FASTA described in Pearson and Lipman Proc. Natl.
Acad.
Sci. 85:2444-8(1988). Within FASTA, ktup is a control option that sets the
sensitivity and
speed of the search.
[0039] The term "derivative" as used herein, refers to a variant polypeptide
of the
invention that comprises, or alternatively consists of, an amino acid sequence
of a BLyS
polypeptide, a fragment of BLyS, or an antibody of the invention that
immunospecifically
binds to BLyS, which has been altered by the introduction of amino acid
residue
substitutions, deletions or additions. The term "derivative" as used herein
also refers to a
BLyS polypeptide, a fragment of BLyS, an antibody that immunospecifically
binds to
BLyS which has been modified, e.g., by the covalent attachment of any type of
molecule
to the polypeptide. For example, but not by way of limitation, a BLyS
polypeptide, a
fragment of BLyS, or an anti-BLyS antibody, may be modified, e.g., by
glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand
or other
protein, etc. A derivative of a BLyS polypeptide, a fragment of BLyS, or an
anti-BLyS
antibody, may be modified by chemical modifications using techniques known to
those of
skill in the art, including, but not limited to, specific chemical cleavage,
acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of
a BLyS
polypeptide, a fragment of BLyS, or an anti-BLyS antibody, may contain one or
more
non-classical amino acids. A polypeptide derivative possesses a similar or
identical
function as a BLyS polypeptide, a fragment of BLyS, or an anti-BLyS antibody,
described
herein.
[0040] The term "epitopes" as used herein refers to portions of BLyS having
antigenic
or immunogenic activity in an animal, preferably a mammal. An epitope having
immunogenic activity is a portion of BLyS that elicits an antibody response in
an animal.
An eptiope having antigenic activity is a portion of BLyS to which an antibody
immunospecifically binds as determined by any method known in the art, for
example, by
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the immunoassays described herein. Antigenic epitopes need not necessarily be
immunogenic.
[0041] The term "fragment" as used herein refers to a polypeptide comprising
an
amino acid sequence of at least 5 amino acid residues, at least 10 amino acid
residues, at
least 15 amino acid residues, at least 20 amino acid residues, at least 25
amino acid
residues, at least 30 amino acid residues, at least 35 amino acid residues, at
least 40 amino
acid residues, at least 45 amino acid residues, at least 50 amino acid
residues, at least 60
amino residues, at least 70 amino acid residues, at least 80 amino acid
residues, at least 90
amino acid residues, at least 100 amino acid residues, at least 125 amino acid
residues, at
least 150 amino acid residues, at least 175 amino acid residues, at least 200
amino acid
residues, or at least 250 amino acid residues, of the amino acid sequence of
BLyS, or an
anti-BLyS antibody (including molecules such as scFv's, that comprise, or
alternatively
consist of, antibody fragments or variants thereof) that immunospecifically
binds to BLyS.
[0042] The term "fusion protein" as used herein refers to a polypeptide that
comprises,
or alternatively consists of, an amino acid sequence of an anti-BLyS antibody
of the
invention and an amino acid sequence of a heterologous polypeptide (i.e., a
polypeptide
unrelated to an antibody or antibody domain).
[0043] The term "host cell" as used herein refers to the particular subject
cell
transfected with a nucleic acid molecule and the progeny or potential progeny
of such a
cell. Progeny may not be identical to the parent cell transfected with the
nucleic acid
molecule due to mutations or environmental influences that may occur in
succeeding
generations or integration of the nucleic acid molecule into the host cell
genome.
[0044] By "isolated antibody" is intended an antibody removed from its native
environment. Thus, an antibody produced andlor contained within a recombinant
host cell
is considered isolated for purposes of the present invention.
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DESCRIPTION OF THE FIGURES
[0045] Figure 1. ELISA results for three scFvs, I006E07, I008D05 and I016F04,
that
immunospecifically bind to U937 membranes, but not to bind to or cross-react
with TNF-
alpha or BSA.
[0046] Figure 2. The results for three scFvs, I016H07, IOO1C09 and I018D07, in
a
receptor inhibition assay.
[0047] Figure 3. ELISA results for two scFvs (I022D01 and I031F02)
demonstrating
their ability to bind to human BLyS and to cross-react with mouse BLyS, but
not to bind
to or cross-react with other antigens of the TNF ligand family.
[0048] Figure 4. ELISA results for three scFvs (I031F09, I050A12, and I051C04)
binding to U937 plasma membranes when either BLyS or TNF-alpha is used as a
competitor.
[0049] Figure 5. Kinetic analysis of scFv antibody I003C02. A dilution series
of
I003C02 from 3nM to 825nM is shown. Association and dissociation curves were
generated using a BIAcore 2000 and BIAevaluation 3.0 software.
[0050] Figure 6. Typical titration curves for two scFv antibodies (I007F11 and
I050A07) are shown in Figure 6. Unlabelled BLyS competed for binding to its
receptor
with an ICSO value of 0.8 nM. The ICso values for I007F11 and I050A07 are 7.9
nM and
17.1 nM , respectively. The assay was performed in triplicate and standard
error bars are
shown.
[0051] Figure 7. ELISA results for three scFvs clones (I074B12, I075F12 and
I075A02) that immunospecifically bind to immobilized BLyS, but not to U937
plasma
membranes, TNF-alpha or BSA. As a control, a phage antibody that recognizes
TNFa, is
also shown in Figure 7.
[0052] Figure 8. The results for two scFvs (I025B09 and I026C04) in a receptor
inhibition assay.
[0053] Figure 9. ELISA results for two scFvs clones (I067F05 and I078D02)
demonstrating their ability to bind to immobilized human BLyS and to cross-
react with
immobilized mouse BLyS, but not to bind to or cross-react with other antigens
of the TNF
ligand family.
[0054] As a control, a phage antibody that recognizes TNFa, is also shown in
Figure
7.
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[0055] Figure 10. Kinetic analysis of scFV antibody I002A01. A dilution series
of
I002A01from 3nM to 1650nM is shown. Association and dissociation curves were
generated using a BIAcore 2000 and BIAevaluation 3.0 software.
[0056] Figure 11. Typical titration curves for two scFvs, I0068C06 and I074B
12, are
shown in Figure 11. Unlabelled BLyS competed for binding to its receptor with
an
inhibitory constant 50 (ICso) value of 0.66 nM. The ICSO values for I0068C06
and I074B 12
are 61 nM and 13 nM , respectively. The assay was performed in triplicate and
standard
error bars are shown.
[0057] Figure 12. ELISA results for three clones (I079C01, I081C10 and
I082A02)
demonstrating their ability to bind histidine-tagged BLyS, U937 plasma
membranes, but
not to bind immobilized biotinylated BLyS.
[0058] Figure 13. ELISA results for three scFvs (I079B04, I079F08, and
I080B01)
binding to U937 plasma membranes when either histidine-tagged BLyS or
biotinylated
BLyS is used as a competitor.
[0059] Figure 14. An example of the dissociation section of a typical
sensorgram for 8
scFvs is shown in Figure 14. An anti-TNFa antibody that does not recognize
BLyS was
included as a control. Of the 8 scFvs exemplified, I079F06 was identified for
further study
due to the relatively high numbers of RU's bound to the surface.
[0060] Figure 15. A typical example of the binding curves generated for the
scFv
antibody I082C03 is shown in Figure 15. The off-rate for this clone was
calculated as
2xlO-3 S 1. The affinity of I082C03 was calculated as 20 nM, assuming 100%
activity of
the scFv.
[0061] Figure 16. ELISA results for three scFvs (I079B04, I079F08, and
I080B01)
binding to P388 plasma membranes when either histidine-tagged BLyS or
biotinylated
BLyS is used as a competitor.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention encompasses antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to BLyS or a fragment or variant of BLyS. In
particular, the
invention provides antibodies such as, for example, single chain Fvs (scFvs)
having an
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WO 03/055979 PCT/US02/36496
amino acid sequence of any one of SEQ ID NOS:1 - 2128, as referred to in Table
1. In
particular, the present invention encompasses antibodies that
immunospecifically bind to a
polypeptide, a polypeptide fragment or variant, or an epitope of human BLyS
(SEQ ID
NOS:3228 and/or 3229) or BLyS expressed on human monocytes; murine BLyS (SEQ
ID
NOS:3230 andlor 3231) or BLyS expressed on murine monocytes; rat BLyS (either
the
soluble forms as given in SEQ ID NOS:3232, 3233, 3234 andlor 3235 or in a
membrane
associated form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g.,
the monkey
BLyS polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey
BLyS,
or BLyS expressed on monkey monocytes) (as determined by immunoassays known in
the
art for assaying specific antibody-antigen binding).
[0063] The polypeptide sequence shown in SEQ ID N0:3228 was obtained by
sequencing and translating the cDNA of the ~DU15 clone which was deposited on
October 22, 1996 at the American Type Culture Collection, 10801 University
Boulevard,
Manassas, Virginia 20110-2209, and assigned ATCC Accession No. 97768. The
deposited clone is contained in the pBluescript SK(-) plasmid (Stratagene, La
Jolla, CA).
The ATCC deposits were made pursuant to the terms of the Budapest Treaty on
the
international recognition of the deposit of microorganisms for the purposes of
patent
procedure.
[0064] The polypeptide sequence shown in SEQ ID N0:3229 was obtained by
sequencing and translating the cDNA of the I~PMC52 clone, which was deposited
on.
December 10, 1998 at the American Type Culture Collection, and assigned ATCC
Accession No. 203518. The deposited clone is contained in the pBluescript SK(-
) plasmid
(Stratagene, La Jolla, CA). The ATCC deposits were made pursuant to the terms
of the
Budapest Treaty on the international recognition of the deposit of
microorganisms for the
purposes of patent procedure.
[0065] The BLyS polypeptides bound by the antibodies of the invention may be
in
monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers).
Accordingly, the present invention relates to antibodies that bind monomers
and multimers
of the BLyS polypeptides of the invention, their preparation, and compositions
(preferably, pharmaceutical compositions) containing them. In specific
embodiments, the
antibodies of the invention bind BLyS monomers, dimers, trimers or tetramers.
In
additional embodiments, the antibodies of the invention bind at least dimers,
at least
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trimers, or at least tetramers of BLyS.
[0066] Multimeric BLyS bound by the antibodies of the invention may be
homomers
or heteromers. A BLyS homomer, refers to a multimer containing only BLyS
polypeptides (including BLyS fragments, variants, and fusion proteins, as
described
herein). These homomers may contain BLyS polypeptides having identical or
different
amino acid sequences. In specific embodiments, the antibodies of the invention
bind a
BLyS homodimer (e.g., containing two BLyS polypeptides having identical or
different
amino acid sequences) or a BLyS homotrimer (e.g., containing three BLyS
polypeptides
having identical or different amino acid sequences). In a preferred
embodiment, the
antibodies of the invention bind homotrimers of BLyS. In additional
embodiments, the
antibodies of the invention bind a homomeric BLyS multimer which is at least a
homodimer, at least a homotrimer, or at least a homotetramer.
[0067] Heteromeric BLyS refers to a multimer containing heterologous
polypeptides
(i.e., polypeptides of a different protein) in addition to the BLyS
polypeptides of the
invention. In a specific embodiment, the antibodies of the invention bind a
BLyS
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the
antibodies of the invention bind a heteromeric BLyS multimer which is at least
a
heterodimer, at least a heterotrimer, or at least a heterotetramer. In highly
preferred
embodiments, the antibodies of the invention bind a heterotrimer comprising
both BLyS
polypeptides and APRIL polypeptides (SEQ m NO:3239; GenBank Accession No.
AF046888; PCT International Publication Number W097/33902; J. Exp. Med.
188(6):1185-1190) or fragments or variants thereof. In other highly preferred
embodiments, the antibodies of the invention bind a heterotrimer comprising
one BLyS
polypeptide (including fragments or variants) and two APRIL polypeptides
(including
fragments or variants). In still other highly preferred embodiments, the
antibodies of the
invention bind a heterotrimer comprising two BLyS polypeptides (including
fragments or
variants) and one APRIL polypeptide (including fragments or variants). In a
further
nonexclusive embodiment, the heteromers bound by the antibodies of the
invention
contain CD40 ligand polypeptide sequence(s), or biologically active fragments)
or
variants) thereof.
[0068] In particularly preferred embodiments, the antibodies of the invention
bind
homomeric, especially homotrimeric, BLyS polypeptides, wherein the individual
protein
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components of the multimers consist of the mature form of BLyS (e.g., amino
acids
residues 134-285 of SEQ ID NO:3228, or amino acids residues 134-266 of SEQ ID
N0:3229) or fragments or variants thereof. In other specific embodiments,
antibodies of
the invention bind heteromeric, especially heterotrimeric, BLyS polypeptides
such as a
heterotrimer containing two BLyS polypeptides and one APRIL polypeptide or a
heterotrimer containing one BLyS polypeptide and two APRIL polypeptides, and
wherein
the individual protein components of the BLyS heteromer consist of the mature
extracellular soluble portion of either BLyS (e.g., amino acids residues 134-
285 of SEQ
ID N0:3228, or amino acids residues 134-266 of SEQ ID N0:3229) or fragments or
variants thereof, or the mature extracellular soluble portion APRIL (e.g.,
amino acid
residues 105-250 of SEQ ID N0:3239) or fragments or variants thereof.
[0069] In specific embodiments, the antibodies of the invention bind
conformational
epitopes of a BLyS monomeric protein. In specific embodiments, the antibodies
of the
invention bind conformational epitopes of a BLyS multimeric, especially
trimeric, protein.
In other embodiments, antibodies of the invention bind conformational epitopes
that arise
from the juxtaposition of BLyS with a heterologous polypeptide, such as might
be present
when BLyS forms heterotrimers (e.g., with APRIL polypeptides (e.g., SEQ ID SEQ
ID
N0:3239)), or in fusion proteins between BLyS and a heterologous polypeptide.
[0070] In a specific embodiment, antibodies of the invention that specifically
bind
heterotrimers containing at least one BLyS polypeptide and at least one APRIL
polypeptide, comprise all or a portion of SEQ ID NOS: 1881 or 1884 (e.g., one
or more
CDR regions, a VH domain or a VL domain). In a specific embodiment, the
heterotrimers
containing at least one BLyS polypeptide and at least one APRIL polypeptide
comprise
two BLyS polypeptides and one APRIL polypeptide. In a specific embodiment, the
heterotrimers containing at least one BLyS polypeptide and at least one APRIL
polypeptide comprise one BLyS polypeptide and two APRIL polypeptides.
[0071] In a specific embodiment, antibodies of the invention that specifically
bind
heterotrimers containing at least one BLyS polypeptide and at least one APRIL
polypeptide, comprise all or a portion of any one of SEQ ID NOS: 3240-3247
(e.g., one or
more CDR regions, a VH domain or a VL domain). The sequences of SEQ ID NOS:
3240-3247 are presented after Table 1 just prior to the claims. In a specific
embodiment,
the heterotrimers containing at least one BLyS polypeptide and at least one
APRIL
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polypeptide comprise two BLyS polypeptides and one APRIL polypeptide. In a
specific
embodiment, the heterotrimers containing at least one BLyS polypeptide and at
least one
APRIL polypeptide comprise one BLyS polypeptide and two APRIL polypeptides.
[0072] BLyS multimers bound by the antibodies of the invention may be the
result of
hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be
indirectly
linked, by for example, liposome formation. Thus, in one embodiment, BLyS
multimers,
such as, for example, homodimers or homotrimers, are formed when polypeptides
of the
invention contact one another in solution. In another embodiment, BLyS
heteromultimers, such as, for example, BLyS heterotrimers or BLyS
heterotetramers, are
formed when polypeptides of the invention contact antibodies to the
polypeptides of the
invention (including antibodies to the heterologous polypeptide sequence in a
fusion
protein of the invention) in solution. In other embodiments, BLyS multimers
are formed
by covalent associations with andlor between the BLyS polypeptides of the
invention.
Such covalent associations may involve one or more amino acid residues
contained in the
polypeptide sequence (e.g., that recited in SEQ ~ NO:3228 or SEQ ID N0:3229).
In one
instance, the covalent associations are cross-linking between cysteine
residues located
within the polypeptide sequences which interact in the native (i.e., naturally
occurring)
polypeptide. In another instance, the covalent associations are the
consequence of
chemical or recombinant manipulation. Alternatively, such covalent
associations may
involve one or more amino acid residues contained in the heterologous
polypeptide
sequence in a BLyS fusion protein. In one example, covalent associations are
between the
heterologous sequence contained in a fusion protein (see, e.g., US Patent
Number
5,478,925). In a specific example, the covalent associations are between the
heterologous
sequence contained in a BLyS-Fc fusion protein. In another specific example,
covalent
associations of fusion proteins of the invention are between heterologous
polypeptide
sequence from another TNF family ligand/receptor member that is capable, of
forming
covalently associated multimers, such as for example, oseteoprotegerin (see,
e.g.,
International Publication No. WO 98/49305, the contents of which are herein
incorporated
by reference in its entirety). In another specific example, covalent
associations of fusion
proteins of the invention are between heterologous polypeptide sequence from
CI~40L, or
a soluble fragment thereof. In another embodiment, two or BLyS polypeptides
are joined
through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer
linkers).
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Examples include those peptide linkers described in U.S. Pat. No. 5,073,627
(hereby
incorporated by reference). Proteins comprising multiple BLyS polypeptides
separated by
peptide linkers may be produced using conventional recombinant DNA technology.
[0073] In one embodiment, antibodies of the invention immunospecifically bind
a
BLyS polypeptide having the amino acid sequence of SEQ ll~ N0:3228 or as
encoded by
the cDNA clone contained in ATCC No. 97768, or a polypeptide comprising a
portion
(i.e., a fragment) of the above polypeptides. In another embodiment, the
invention
provides an antibody that binds an isolated BLyS polypeptide having the amino
acid
sequence of SEQ ID NO:3229 or the amino acid sequence encoded by the cDNA
clone
contained in ATCC No. 203518, or an antibody that binds polypeptide comprising
a
portion (i.e, fragment) of the above polypeptides.
[0074] Antibodies of the invention that bind BLyS polypeptides may bind them
in as
isolated polypeptides, in their naturally occurring state and/or their native
conformation.
By "isolated polypeptide" is intended a polypeptide removed from its native
environment.
Thus, a polypeptide produced by and/or contained within a recombinant host
cell is
considered isolated for purposes of the present invention. Also intended as an
"isolated
polypeptide" are polypeptides that have been purified, partially or
substantially, from a
recombinant host cell. Thus, antibodies of the present invention may bind
recombinantly
produced BLyS polypeptides.
[0075] Antibodies of the present invention may also bind BLyS expressed on the
surface of a cell, wherein said BLyS polypeptide is encoded by a
polynucleotide encoding
amino acids 1 to 285 of SEQ ID N0:2 operably associated with a regulatory
sequence that
controls expression of said polynucleotide. In certain embodiments, said BLyS
polypeptide expressed on the surface of a cell is a recombinant BLyS
polypeptide. In
other embodiments, said BLyS polypeptide expressed on the surface of the cell
is a
naturally occurring BLyS polypeptide. As a non-limiting example, an antibody
of the
invention may bind a BLyS expressed on the surface of the cell wherein Lys-132
and/or
Arg-133 of the BLyS sequence shown in SEQ ID N0:3228 is mutated to another
amino
acid residue, or deleted altogether, thereby preventing or diminishing release
of the soluble
form of BLyS from cells expressing BLyS.
[0076] Antibodies of the present invention may also bind BLyS secreted by a
cell,
wherein said BLyS polypeptide is encoded by a polynucleotide encoding amino
acids 1 to
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WO 03/055979 PCT/US02/36496
285 of SEQ ID NO:2 operably associated with a regulatory sequence that
controls
expression of said polynucleotide. In certain embodiments, said BLyS
polypeptide
secreted by a cell is a recombinant BLyS polypeptide. In other embodiments,
said BLyS
polypeptide secreted by a cell is a naturally occurnng BLyS polypeptide.
[0077] Antibodies of the present invention immunospecifically bind to
polypeptides
comprising or alternatively, consisting of, the amino acid sequence of SEQ ID
N0:3228,
encoded by the cDNA contained in the plasmid having ATCC accession number
97768, or
encoded by nucleic acids which hybridize (e.g., under stringent hybridization
conditions)
to the nucleotide sequence contained in the deposited clone. Antibodies of the
present
invention also bind to fragments of the amino acid sequence of SEQ ID N0:3228,
encoded by the cDNA contained in the plasmid having ATCC accession number
97768, or
encoded by nucleic acids which hybridize (e.g., under stringent hybridization
conditions)
to the nucleotide sequence contained in the deposited clone.
[0078] Additionally, antibodies of the present invention bind polypeptides
comprising
or alternatively, consisting of, the amino acid sequence of SEQ ID N0:3229,
encoded by
the cDNA contained in the plasmid having ATCC accession number 203518, or
encoded
by nucleic acids which hybridize (e.g., under stringent hybridization
conditions) to the
nucleotide sequence contained in the deposited clone. Antibodies of the
present invention
also bind to fragments of the amino acid sequence of SEQ ID N0:3229, encoded
by the
cDNA contained in the plasmid having ATCC accession number 203518, or encoded
by
nucleic acids which hybridize (e.g., under stringent hybridization conditions)
to the
nucleotide sequence contained in the deposited clone.
[0079] In addition, antibodies of the invention bind polypeptides or
polypeptide
fragments comprising or alternatively, consisting of, an amino acid sequence
contained in
SEQ >D NOS: 3230 through 3237.
[0080] In specific embodiments, the antibodies of the present invention
immunospecifically bind polypeptide fragments including polypeptides
comprising or
alternatively, consisting of, an amino acid sequence contained in SEQ ll~
N0:3228,
encoded by the cDNA contained in the deposited clone, or encoded by nucleic
acids which
hybridize (e.g., under stringent hybridization conditions) to the nucleotide
sequence
contained in the deposited clone. Protein fragments may be "free-standing," or
comprised
within a larger polypeptide of which the fragment forms a part or region, most
preferably
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as a single continuous region. Representative examples of polypeptide
fragments that may
be bound by the antibodies of the present invention, include, for example,
fragments that
comprise or alternatively, consist of from about amino acid residues: 1 to 50,
51 to 100,
101 to 150, 151 to 200, 201 to 250, and/or 251 to 285 of SEQ ID N0:3228.
Moreover,
polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130,
140, 150, 175 or 200 amino acids in length.
[0081] In specific embodiments, antibodies of the present invention bind
polypeptide
fragments comprising, or alternatively consisting of, amino acid residues: 1-
46, 31-44,
47-72, 73-285, 73-83, 94-102, 148-152, 166-181, 185-209, 210-221, 226-237, 244-
249,
253-265, and/or 277-285 of SEQ ID N0:3228. In a specific embodiment,
antibodies of the
invention bind an epitope comprising amino acids 165-171 of SEQ ID N0:3228. In
another embodiment, the CDRs of antibodies of the invention make contacts with
one or
more amino acids in the the sequence of amino acids 165-171 of SEQ ID N0:3228.
In
another embodiment, antibodies of the invention whose CDRs make contact with
one or
more amino acids in the the sequence of amino acids 165-171 of SEQ ID N0:3228
disrupt
BLyS- BLyS receptor interactions.
[0082] It will be recognized by one of ordinary skill in the art that
mutations targeted
to regions of a BLyS polypeptide of SEQ ID N0:3228 which encompass the
nineteen
amino acid residue insertion which is not found in the BLyS polypeptide
sequence of SEQ
ID N0:3229 (i.e., amino acid residues Val-142 through Lys-160 of the sequence
of SEQ
ID N0:3229) may affect the observed biological activities of the BLyS
polypeptide. More
specifically, a partial, non-limiting and non-exclusive list of such residues
of the BLyS
polypeptide sequence which may be targeted for mutation includes the following
amino
acid residues of the BLyS polypeptide sequence as shown in SEQ ID N0:3228: V-
142;
T-143; Q-144; D-145; C-146; L-147; Q-148; L-149; I-150; A-151; D-152; S-153; E-
154;
T-155; P-156; T-157; I-158; Q-159; and K-160. Thus, in specific embodiments,
antibodies
of the present invention that bind BLyS polypeptides which have one or more
mutations in
the region from V-142 through K-160 of SEQ ID N0:3228 are contemplated.
[0083] Polypeptide fragments may be "free-standing," or comprised within a
larger
polypeptide of which the fragment forms a part or region, most preferably as a
single
continuous region. Representative examples of polypeptide fragments that may
be bound
by antibodies of the present invention, include, for example, fragments that
comprise or
CA 02467521 2004-05-14
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alternatively, consist of from about amino acid residues: 1 to 15, 16-30, 31-
46, 47-55, 56-
72, 73-104, 105-163, 163-188, 186-210 and 210-284 of the amino acid sequence
disclosed
in SEQ 1D N0:3228. Additional representative examples of polypeptide fragments
that
may be bound by antibodies of the present invention, include, for example,
fragments that
comprise or alternatively, consist of from about amino acid residues: 1 to
143, 1-150, 47-
143, 47-150, 73-143, 73-150, 100-150, 140-145, 142-148, 140-150, 140-200, 140-
225,
and 140-266 of the amino acid sequence disclosed in SEQ )D N0:3229. Moreover,
polypeptide fragments that may be bound by antibodies of the present
invention, can be at
least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or
200 amino
acids in length. In this context, "about" means the particularly recited
ranges and ranges
larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at
either or both the
amino- and carboxy-termini.
[0084] Additional preferred embodiments encompass antibodies that bind
polypeptide
fragments comprising, or alternatively consisting of, the predicted
intracellular domain of
BLyS (e.g., amino acid residues 1-46 of SEQ ID N0:3228), the predicted
transmembrane
domain of BLyS (e.g., amino acid residues 47-72 of SEQ ID N0:3228), the
predicted
extracellular domain of BLyS (e.g., amino acid residues 73-285 of SEQ )D
N0:3228), the
mature soluble extracellular domain of BLyS (e.g., amino acids residues 134-
285 of SEQ
1D N0:3228), the predicted TNF conserved domain of BLyS (e.g., amino acids 191
to 284
of SEQ m N0:3228), and a polypeptide comprising, or alternatively, consisting
of the
predicted intracellular domain fused to the predicted extracellular domain of
BLyS (amino
acid residues 1-46 fused to amino acid residues 73-285 of SEQ ID N0:3228).
[0085] Further additional preferred embodiments encompass polypeptide
fragments
comprising, or alternatively consisting of, the predicted intracellular domain
of BLyS
(amino acid residues 1-46 of SEQ )D N0:3229), the predicted transmembrane
domain of
BLyS (amino acid residues 47-72 of SEQ 1D N0:3229), the predicted
extracellular
domain of BLyS (amino acid residues 73-266 of SEQ )D N0:3229), the predicted
TNF
conserved domain of BLyS (amino acids 172 to 265 of SEQ ID N0:3229), and a
polypeptide comprising, or alternatively, consisting of the predicted
intracellular domain
fused to the predicted extracellular domain of BLyS (amino acid residues 1-46
fused to
amino acid residues 73-266 of SEQ ID N0:3229).
[0086] Certain additional embodiments of the invention encompass antibodies
that
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bind polypeptide fragments comprising, or alternatively consisting of, the
predicted beta-
pleated sheet regions of the BLyS polypeptides of SEQ ID N0:3228 and SEQ ID
N0:3229. These polypeptide fragments comprising the beta-pleated sheets of
BLyS
comprise, or alternatively consist of, amino acid residues Gln-144 to Ala-151,
Phe-172 to
Lys-173, Ala-177 to Glu-179, Asn-183 to Ile-185, Gly-191 to Lys-204, His-210
to Val-
219, Leu-226 to Pro-237, Asn-242 to Ala-251, Gly-256 to Ile-263 and/or Val-276
to Leu-
284 of SEQ ID N0:3228. In another, nonexclusive embodiment, these polypeptide
fragments comprising the beta-pleated sheets of BLyS comprise, or
alternatively consist
of, amino acid residues Phe-153 to Lys-154, Ala-158 to Glu-160, Asn-164 to Ile-
166, Gly-
172 to Lys-185, His-191 to Val-200, Leu-207 to Pro-218, Asn-223 to Ala-232,
Gly-237 to
Ile-244 and/or Val-257 to Leu-265 of SEQ ID NO:3229.
[0087] A partial, non-limiting, and exemplary list of polypeptides that may be
bound
by the antibodies of the invention includes polypeptides that comprise, or
alternatively
consist of, combinations of amino acid sequences of the invention includes,
for example,
[Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142 to Lys-160]
fused to
[Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-
285] of SEQ
ID NO:3228; or [Met-1 to Lys-113] fused to [Val-142 to Lys-160] fused to [Gly-
161 to
Gln-198] fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-285] of SEQ ID
N0:3228; or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142
to Lys-
160] fused to [Gly-161 to Gln-198] fused to [Gly-249 to Leu-285] of SEQ ID
N0:3228.
Other combinations of amino acids sequences that may be bound by the
antibodies of the
invention may include the polypeptide fragments in an order other than that
recited above
(e.g., [Leu-114 to Thr-141] fused to [Val-199 to Ala-248] fused to [Gly-249 to
Leu-285]
fused to [Val-142 to Lys-160] of (SEQ ID N0:3228). Other combinations of amino
acids
sequences that may be bound by the antibodies of the invention may also
include
heterologous polypeptide fragments as described herein and/or other
polypeptides or
polypeptide fragments of the present invention (e.g., [Met-1 to Lys-113] fused
to [Leu-114
to Thr-141] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused
to [Gly-
249 to Leu-285] of SEQ ID N0:3228 fused to a FLAG tag ; or [Met-1 to Lys-113]
of SEQ
ID N0:3228 fused to [Leu-114 to Thr-141] of SEQ ID N0:3228 fused to [Glu-135
to
Asn-165] of SEQ ID N0:39 fused to [Val-142 to Lys-160] of SEQ ID N0:3228 fused
to
[Gly-161 to Gln-198] of SEQ ID N0:3228 fused to [Val-199 to Ala-248] of SEQ ID
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N0:3228 fused to [Gly-249 to Leu-285] of SEQ ID N0:3228).
[0088] A partial, non-limiting, and exemplary list of polypeptides that may be
bound
by the antibodies of the invention includes polypeptides that comprise, or
alternatively
consist of, combinations of amino acid sequences includes, for example, [Met-1
to Lys-
113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Val-
180 to
Ala-229] fused to [Gly-230 to Leu-266] of SEQ ID N0:3229; [Met-1 to Lys-113]
fused to
[Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-
266] of SEQ
ID N0:3229; or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-
142 to
Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID N0:3229. Other of amino acids
sequences that may be bound by the antibodies of the invention combinations
may include
the polypeptide fragments in an order other than that recited above (e.g.,
[Leu-114 to Thr-
141] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] fused to [Gly-
142 to
Gln-179] of SEQ ID NO:3229). Other combinations of amino acid sequences that
may be
bound by the antibodies of the invention may also include heterologous
polypeptide
fragments as described herein and/or other polypeptides or polypeptide
fragments of the
present invention (e.g., [Met-1 to Lys-113] fused to [Leu-114 to Thr-141]
fused to [Gly-
142 to Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID N0:3229 fused to a
FLAG tag
(SEQ ID N0:3238) or, [Met-1 to Lys-113] of SEQ ID NO:3229 fused to [Leu-114 to
Thr-
141] of SEQ ID NO:3229 fused to [Glu-135 to Asn-165] of SEQ ID N0:39 fused to
[Gly-
142 to Gln-179] of SEQ ID N0:3229 fused to [Val-180 to Ala-229] of SEQ ID
N0:3229
fused to [Gly-230 to Leu-266] of SEQ ID N0:3229.
[0089] Additional embodiments of the invention encompass antibodies that bind
BLyS
polypeptide fragments comprising, or alternatively consisting of, functional
regions of
polypeptides of the invention, such as the Gamier-Robson alpha-regions, beta-
regions,
turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and
coil-regions,
Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha-
and
beta-amphipathic regions, Karplus-Schulz flexible regions, Emini surface-
forming regions
and Jameson-Wolf regions of high antigenic index set out in Tables 9 and 10
and as
described herein. In a preferred embodiment, the polypeptide fragments bound
by the
antibodies of the invention are antigenic (i.e., containing four or more
contiguous amino
acids having an antigenic index of greater than or equal to 1.5, as identified
using the
default parameters of the Jameson-Wolf program) of a complete (i.e., full-
length) BLyS
33
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WO 03/055979 PCT/US02/36496
polypeptide (e.g., SEQ ID NOS:3228 and 3229).
[0090] The data representing the structural or functional attributes of the
BLyS
polypeptide of SEQ ID NO:3228 (Table 9) or the BLyS polypeptide of SEQ ID
NO:3229
(Table 10), as described above, was generated using the various modules and
algorithms
of the DNA*STAR set on default parameters. Column I represents the results of
a
Gamier-Robson analysis of alpha helical regions; Column II represents the
results of a
Chou-Fasman analysis of alpha helical regions; Column III represents the
results of a
Gamier Robson analysis of beta sheet regions; Column IV represents the results
of a
Chou-Fasman analysis of beta sheet regions; Column V represents the results of
a Gamier
Robson analysis of turn regions; Column VI represents the results of a Chou-
Fasman
analysis of turn regions; Column VII represents the results of a Gamier Robson
analysis of
coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot;
Column IX
represents a Hopp-Woods hydrophobicity plot; Column X represents the results
of an
Eisenberg analysis of alpha amphipathic regions; Column XI represents the
results of an
Eisenberg analysis of beta amphipathic regions; Column XII represents the
results of a
Karplus-Schultz analysis of flexible regions; Column XIII represents the
Jameson-Wolf
antigenic index score; and Column XIV represents the Emini surface probability
plot.
[0091] In a preferred embodiment, the data presented in columns VIII, IX,
XITI, and
XIV of Tables 9 and 10 can be used to determine regions of the BLyS
polypeptide of SEQ
ID N0:3228 (Table 9) or the BLyS polypeptide of SEQ ID N0:3229 (Table 10)
which
exhibit a high degree of potential for antigenicity. Regions of high
antigenicity are
determined from the data presented in columns VIII, IX, XIII, and/or XIV by
choosing
values which represent regions of the polypeptide which are likely to be
exposed on the
surface of the polypeptide in an environment in which antigen recognition may
occur in
the process of initiation of an immune response.
[0092] The above-mentioned preferred regions set out in Tables 9 and 10
include, but
are not limited to, regions of the aforementioned types identified by analysis
of the amino
acid sequence set out in SEQ ID NO:2. As set out in Tables 9 and 10, such
preferred
regions include Gamier-Robson alpha-regions, beta-regions, turn-regions, and
coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-
Doolittle
hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-
Schulz
flexible regions, Jameson-Wolf regions of high antigenic index and Emini
surface-forming
34
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WO 03/055979 PCT/US02/36496
regions. Preferably, antibodies of the present invention bind BLyS
polypeptides or BLyS
polypeptide fragments and variants comprising regions of BLyS that combine
several
structural features, such as several (e.g., 1, 2, 3 , or 4) of the same or
different region
features set out above and in Tables 9 and 10.
CA 02467521 2004-05-14
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Table '
9
Res Position I II III IV V VI VII VIII IX X XI XIIXIII XIV
Met 1 A . . . . . . 0.73 -0.71. . . 0.95 1.39
Asp 2 A . . . . T . 1.12 -0.66* . . 1.15 1.56
Asp 3 A . . . . T . 1.62 -1.09* . . 1.15 2.12
Ser 4 A . . . . T . 2.01 -1.51. . . 1.15 4.19
Thr 5 A . . . . T . 2.40 -2.13. . F 1.30 4.35
Glu 6 A A . . . . . 2.70 -1.73* * F 0.90 4.51
Arg 7 A A . . . . . 2.81 -1.34* * F 0.90 4.51
Glu 8 A A . . . . . 2.00 -1.73* * F 0.90 6.12
Gln 9 A A . . . . . 1.99 -1.53* * F 0.90 2.91
Ser 10 A . . B . . . 2.00 -1.04* * F 0.90 2.15
Arg 11 A . . B . . . 1.33 -0.66* * F 0.90 1.66
Leu 12 A . . B . . . 0.41 -0.09* * F 0.45 0.51
Thr 13 A . . B . . . 0.46 0.20 * * F -0.150.32
Ser 14 A A . . . . . 0.50 -0.19* * . 0.30 0.32
Cys 15 A A . . . . . 0.91 -0.19* * . 0.30 0.78
Leu 16 A A . . . . . 0.80 -0.87* * F 0.90 1.06
Lys 17 A A . . . . . 1.61 -1.36. * F 0.90 1.37
Lys 18 A A . . . . . 1.32 -1.74. * F 0.90 4.44
Arg 19 A A . . . . . 1.67 -1.70. * F 0.90 5.33
Glu 20 A A . . . . . 1.52 -2.39. * F 0.90 5.33
Glu 21 A A . . . . . 2.38 -1.70. * F 0.90 2.20
Met 22 A A . . . . . 2.33 -1.70. * F 0.90 2.24
Lys 23 A A . . . . . 1.62 -1.70* * F 0.90 2.24
Leu 24 A A . . . . . 0.66 -1.13* * F 0.75 0.69
Lys 25 A A . . . . . 0.36 -0.49. * F 0.45 0.52
Glu 26 A A . B . . . -0.53-0.71* * . 0.60 0.35
Cys 27 A A . B . . . -0.74-0.03* * . 0.30 0.30
Val 28 A A . B . . . -1.00-0.03* * . 0.30 0.12
Ser 29 A A . B . . . -0.080.40 * * . -0.300.11
Ile 30 A . . B . . . -0.080.40 * * . -0.300.40
Leu 31 A . . B . . . -0.08-0.17* . . 0.45 1.08
Pro 32 . . . B . . C 0.29 -0.81* . F 1.10 1.39
Arg 33 . . . . T . . 0.93 -0.81. * F 1.50 2.66
Lys 34 . . . . T . . 0.93 -1.07. . F 1.84 4.98
Glu 35 . . . . . . C 0.97 -1.37* * F 1.98 4.32
_ Ser 36 _ . . , . T C 1.89 _1.16. * F 2.52 1.64
. *. _
Pro 37 . . . . . T C 1.80 -1.16* * F 2.86 1.60
Ser 38 . . . . T T . 1.39 -0.77* . F 3.40 1.24
Val 39 A . . . . T . 1.39 -0.39. * F 2.36 1.24
Arg 40 A . . . . . . 1.39 -0.77* * F 2.46 1.60
Ser 41 A . . . . . . 1.34 -1.20* * F 2.46 2.00
Ser 42 . . . . T T . 1.60 -1.16. * F 3.06 2.67
Lys 43 . . . . T T . 1.09 -1.80. * F 3.06 2.72
Asp 44 . . . . T T . 1.13 -1.11* * F 3.40 1.67
Gly 45 A . . . . T . 0.43 -0.81* * F 2.66 1.03
Lys 46 A A . . . . . 0.14 -0.70. . F 1.77 0.52
Leu 47 A A . . . . . 0.13 -0.20* . . 0.98 0.31
Leu 48 A A . . . . . -0.720.29 * . . 0.04 0.46
Ala 49 A A . . . . . -1.530.54 . * . -0.600.19
Ala 50 A A . . . . . -2.001.23 . . . -0.600.19
36
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Table
9 (continued)
Res Position I II III IV V VI VII VIII IX X XI XIIXIII XIV
Thr 51 A A . . . . . -2.63 1.23 . . -0.600.19
.
Leu 52 A A . . . . . -2.63 1.04 . . -0.600.19
.
Leu 53 A A . . . . . -2.63 1.23 . . -0.600.15
.
Leu 54 A A . . . . . -2.34 1.41 . . -0.600.09
.
Ala 55 A A . . . . . -2.42 1.31 . . -0.600.14
.
Leu 56 A A . . . . . -2.78 1.20 . . -0.600.09
.
Leu 57 A . . . . T . -2.78 1.09 . . -0.200.06
.
Ser 58 A . . . . T . -2.28 1.09 . . -0.200.05
.
Cys 59 A . . . . T . -2.32 1.07 . . -0.200.09
.
Cys 60 A . . . . T . -2.59 1.03 . . -0.200.08
.
Leu 61 . . B B . . . -2.08 0.99 . . -0.600.04
.
Thr 62 . . B B . . . -1.97 0.99 . . -0.600.11
.
Val 63 . . B B . . . -1.91 1.20 . . -0.600.17
.
Val 64 . . B B . . . -1.24 1.39 . . -0.600.33
.
Ser 65 . . B B . . . -1.43 1.10 . . -0.600.40
.
Phe 66 A . . B . . . -1.21 1.26 . . -0.600.40
.
Tyr 67 A . . B . . . -1.49 1.11 . . -0.600.54
.
Gln 68 A . . B . . . -1.44 0.97 . . -0.600.41
.
Val 69 A . . B . . . -0.59 1.27 . . -0.600.39
.
Ala 70 A . . B . . . -0.63 0.89 . . -0.600.43
.
Ala 71 A . . B . . . 0.07 0.56 * . -0.600.25
.
Leu 72 A . . . . T . -0.50 0.16 * . 0.10 0.55
.
Gln 73 A . . . . T . -1.09 0.20 . F 0.25 0.45
.
Gly 74 A . . . . T . -0.53 0.20 . F 0.25 0.45
.
Asp 75 A . . . . T . -0.76 0.09 * F 0.25 0.73
.
Leu 76 A A . . . . . -0.06 0.09 * F -0.150.35
.
Ala 77 A A . . . . . 0.17 -0.31 * . 0.30 0.69
.
Ser 78 A A . . . . . 0.17 -0.24 * . 0.30 0.42
.
Leu 79 A A . . . . . -0.30 -0.24 * . 0.30 0.88
.
Arg 80 A A . . . . . -0.30 -0.24 * . 0.30 0.72
.
Ala 81 A A . . . . . 0.17 -0.34 * . 0.30 0.93
.
Glu 82 A A . . . . . 0.72 -0.30 * . 0.45 1.11
.
Leu 83 A A . . . . . 0.99 -0.49 * . 0.30 0.77
.
Gln 84 A A . . . . . 1.21 0.01 * . -0.151.04
.
Gly 85 A A . . . . . 1.10 0.01 * . -0.300.61
*
_. _-_ 86 A A , _ _ , , 1.73 0.01 ~ . -0.15_
___ His - - . -- _- ~ _1.27
His 87 A A . . . . . 0.92 -0.67 * . 0.75 1.47
.
Ala 88 A A . . . . . 1.52 -0.39 * . 0.45 1.22
.
Glu 89 A A . . . . . 0.93 -0.39 . . 0.45 1.39
.
Lys 90 A A . . . . . 0.93 -0.39 . F 0.60 1.03
*
Leu 91 A . . . . T . 0.38 -0.46 . . 0.85 1.01
*
Pro 92 A . . . . T . 0.07 -0.46 . . 0.70 0.59
.
Ala 93 A . . . . T . 0.07 -0.03 . . 0.70 0.29
.
Gly 94 A . . . . T . -0.14 0.47 . . -0.200.36
.
Ala 95 A . . . . . . -0.14 0.21 * . -0.100.36
.
Gly 96 A . . . . . . 0.08 -0.21 . F 0.65 0.71
.
Ala 97 A . . . . . . -0.06 -0.21 . F 0.65 0.72
.
Pro 98 A . . . . . . -0.28 -0.21 * F 0.65 0.71
.
Lys 99 A A . . . . . 0.07 -0.03 . F 0.45 0.59
.
Ala 100 A A . . . . . 0.66 -0.46 . F 0.60 1.01
.
37
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Table 9 (continued)
Res I II IIIIV V VI VII VIII IX X XI XII XIIIXIV
Position
Gly 101 A A . . . . . 0.41 -0.96. . F 0.901.13
Leu 102 A A . . . . . 0.79 -0.89. . F 0.750.57
Glu 103 A A . . . . . 0.41 -0.46* . F 0.450.88
Glu 104 A A . . . . . -0.49-0.46* . F 0.450.89
Ala 105 A A . . . . . -0.21-0.24. . . 0.300.81
Pro 106 A A . . . . . -0.46-0.44. . . 0.300.67
Ala 107 A A . . . . . 0.01 0.06 . . . -0.300.39
Val 108 A A . . . . . -0.800.49 . * . -0.600.38
Thr 109 A A . . . . . -0.760.67 . * . -0.600.20
Ala 110 A A . . . . . -1.060.24 * * . -0.300.40
Gly 111 A A . . . . . -1.540.43 * * . -0.600.38
Leu 112 A A . . . . . -0.960.57 * * . -0.600.23
Lys 113 . A B . . . . -0.310.09 * * . -0.300.39
Ile 114 . A B . . . . -0.210.01 * . . -0.300.61
Phe 115 . A B . . . . -0.210.01 * . . 0.151.15
Glu 116 A . . . . C -0.08-0.17* . F 1.250.58
Pro 117 . A . . . . C 0.39 0.26 * * F 1.101.28
Pro 118 . . . . . ~ . C 0.34 -0.00. . F 2.201.47
Ala 119 . . . . . T C 0.89 -0.79. * F 3.001.47
Pro 120 . . . . . T C 1.59 -0.36. * F 2.250.94
Gly 121 . . . . T T . 1.29 -0.39. * F 2.150.98
Glu 122 . . . . T T . 1.20 -0.43. . F 2.001.30
Gly 123 . . . . . . C 1.41 -0.54. . F 1.601.12
Asn 124 . . . . . T C 2.00 -0.57. . F 1.501.97
Ser 125 . . . . . T C 1.91 -0.60. * F 1.501.82
Ser 126 . . . . . T C 2.37 -0.21. * F 1.542.47
Gln 127 . . . . . T C 2.37 -0.64. * F 2.183.01
Asn 128 . . . . . . C 2.76 -0.64. . F 2.323.61
Ser 129 . . . . . T C 2.87 -1.03. . F 2.865.39
Arg 130 . . . . T T 2.58 -1.41* . F 3.406.09
Asn 131 . . . . T T . 2.02 -1.31* . F 3.063.83
Lys 132 . . . . T T . 2.02 -1.07* . F 2.722.12
Arg 133 . . . . T . . 1.68 -1.06* . F 2.181.88
Ala 134 . . . . . . C 1.77 -0.63* . F 1.641.15
Val 135 . . . . . . C 1.66 -0.60* . F 1.49'0.89
Gln 136 . . . , _ . , C. _ -0.60. , F _ 0.79
~ , _ _ * 1.83
1.66
Gly 137 . . . . . T C 1.30 -0.60* . F 2.521.35
Pro 138 . . . . . T C 0.33 -0.61* . F 2.862.63
Glu 139 . . . . T T . 0.61 -0.61* . F 3.401.13
Glu 140 A . . . . T . 1.47 -0.53* . F 2.661.64
Thr 141 A . . . . . . 1.47 -0.56. . F 2.121.84
Val 142 A . . . . . . 1.14 -0.99. . F 1.781.77
Thr 143 A . . . . T . 0.54 -0.41. . F 1.190.55
Gln 144 A . . . . T . 0.54 0.27 * . F 0.250.31
Asp 145 A . . . . T . -0.270.19 * . F 0.250.73
Cys 146 A . . . . T . -0.840.23 * . . 0.100.42
Leu 147 A A . . . . . -0.580.43 * . . -0.600.17
Gln 148 A A . . . . . -0.270.53 * . . -0.600.10
Leu 149 A A . . . . . -0.570.53 * * : -0.300.32
Ile 150 A A . . . . . -0.570.34 * . . 0.300.52
38
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Table 9 (continued)
Res I II IIIIV V VI VII VIII IX X XI XIIXIIIXIV
Position
Ala 151 . A . . . . C -0.21-0.34. * . 1.400.52
Asp 152 . . . . T T . 0.39 -0.26. * F 2.450.91
5er 153 . . . . . T C 0.08 -0.51. . F 3.002.00
Glu 154 . . . . . T C -0.00-0.71. . F 2.702.86
Thr 155 . . . . . T C 0.89 -0.53* . F 2.401.20
Pro 156 . . . B . . C 1.52 -0.13* . F 1.561.55
Thr 157 . . . B T . . 1.18 -0.51* . F 1.921.79
Ile 158 A . . B . . . 1.18 -0.09. . F 1.081.23
Gln 159 . . . . T T . 0.93 -0.19. . F 2.041.07
Lys 160 . . . . T T . 0.93 0.14* . F 1.601.16
Gly 161 . . . . T T . 0.44 0.14* . F 1.442.38
Ser 162 . . . . T T . -0.100.24* . F 1.281.19
Tyr 163 . . . B T . . 0.58 0.49* . . 0.120.44
Thr 164 . . B B . . . 0.29 0.91* . . -0.440.69
Phe 165 . . B B . . . -0.571.40* . . -0.600.54
Val 166 . . B B . . . -1.031.70. . . -0.600.29
Pro 167 . . B B . . . -1.031.63. . . -0.600.16
Trp 168 A . . B . . . -1.491.53. * . -0.600.25
Leu 169 A . . B . . . -1.131.53* . . -0.600.29
Leu 170 A . . B . . . -0.320.89* . . -0.300.38
Ter 171 A . . . . . . 0.19 0.46* . . 0.200.71
Phe 172 . . . . T . . 0.10 -0.03* . . 1.800.85
Lys 173 . . . . T T . -0.20-0.33* . F 2.601.38
Arg 174 . . . . . T C -0.20-0.51. . F 3.001.04
Gly 175 . . . . . T C 0.61 -0.21. . F 2.250.99
Ser 176 A . . . . T . 0.91 -1.00* . F 2.050.86
Ala 177 A A . . . . . 1.66 -1.00* . F 1.350.76
Leu 178 A A . . . . . 1.61 -1.00. . F 1.201.54
Glu 179 A A . . . . . 1.50 -1.43. . F 0.901.98
Glu 180 A A . . . . . 1.89 -1.41* . F 0.903.16
Lys 181 A A . . . . . 1.30 -1.91* . F 0.907.66
Glu 182 A A . . . . . 1.08 -1.91. . F 0.903.10
Asn 183 A A . . . . . 1.03 -1.23* * F 0.901.48
Lys 184 A A . . . . . 1.08 -0.59* . F 0.750.55
Ile 185 A A . . . . . 1.08 -0.59* * . 0.600.63
Leu 1-86 A A . - . . . 0.72 -0.59* *- . 0.60--0.68
-- . -- -
Val 187 A A . . . . . 0.38 -0.50. * . 0.300.49
Lys 188 A A . . . . . 0.13 -0.07* * F 0.450.69
Glu 189 A . . . . T . -0.610.00* * F 0.401.32
Thr 190 . . . . T T . -0.420.10. * F 0.801.54
Gly 191 . . . . T T . -0.500.24* . F 0.650.67
Tyr 192 . . . . T T . 0.11 0.93* * . 0.200.27
Phe 193 . . B B . . . -0.281.69. . . -0.600.29
Phe 194 . . B B . . . -0.281.63. * . -0.600.29
Ile 195 . . B B . . . -0.821.60. . . -0.600.32
Tyr 196 . . B B . . . -1.291.49. . . -0.600.28
Gly 197 . . . B T . . -1.291.39. . . -0.200.26
Gln 198 . . . B T . . -0.901.36. . . -0.200.59
V 199 . . . B . . C -0.201.16. . . -0.400.54
al
Leu 200 . . . B . . C 0.73 0.40. . . -0.100.92
39
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Table 9 (continued)
Res I II III IV V VI VII VIII IX X XI XII XIIIXIV
Position
Tyr 201 . . . . T T . 0.67 -0.03. . . 1.251.06
Thr 202 . . . . T T . 0.77 0.06 . . F 0.802.06
Asp 203 . . . . T T . 0.18 0.17 . . F 0.803.91
Lys 204 A . . . . T . 0.43 -0.01. . F 1.002.52
Thr 205 A A . . . . . 0.90 -0.16. . F 0.601.73
Tyr 206 A A . . . . . 1.11 -0.21. . . 0.451.03
Ala 207 A A . . . . . 0,61 0.29 . . . -0.300.70
Met 208 A A . . . . . -0.280.97 . . . -0.600.40
Gly 209 A A . B . . . -0.321.17 * . . -0.600.18
His 210 A A . B . . . 0.10 0.81 * . . -0.600.31
Leu 211 A A . B . . . 0.39 0.31 . . . -0.300.61
Ile 212 A A . B . . . 1.02 -0.30. . . 0.451.22
Gln 213 A A . B . . . 0.77 -0.73. * . 0.751.80
Arg 214 A A . B . . . 1.08 -0.59. * F 0.901.62
Lys 215 A A . B . . . 0.26 -0.77* * F 0.903.14
Lys 216 A A . B . . . 0.37 -0.81. * F 0.901.35
Val 217 . A B B . . . 0.91 -0.43* * . 0.300.60
His 218 . A B B . . . 0.91 -0.00. * . 0.300.29
Val 219 . A B B . . . 0.80 -0.00* * . 0.300.25
Phe 220 . . B B . . . -0.06-0.00* . . 0.300.57
Gly 221 A . . B . . . -0.400.04 . * . -0.300.35
Asp 222 A . . . . . . -0.36-0.07* . . 0.500.63
Glu 223 A . . . . . . -1.18-0.03* . . 0.500.60
Leu 224 A . . B . . . -0.63-0.17. . . 0.300.45
Ser 225 A . . B . . . -0.74-0.11. . . 0.300.39
Leu 226 A . . B . . . -1.100.57 . * . -0.600.18
Val 227 A . . B . . . -0.991.36 . * . -0.600.19
Thr 228 A . . B . . . -1.660.67 * * . -0.600.28
Leu 229 A . . B . . . -1.730.86 * . . -0.600.18
Phe 230 A . . B . . . -1.430.86 * . . -0.600.17
Arg 231 A . . B . . . -0.620.61 * . . -0.600.21
Cys 232 . . . B T . . -0.370.53 * . . -0.200.41
Ile 233 . . . B T . . -0.270.46 * . . -0.200.46
Gln 234 . . . B T . . 0.54 0.10 * . . 0.100.37
Asn 235 . . . B . . C 0.93 0.10 * . . 0.051.19
Met 236- . . . B . . C- - 0.01 - . - 0.20-2.44
- 0.01--- * F -
Pro 237. . . . B . . C 0.47 0.01 * . F 0.441.16
Glu 238 . . . . T . . 1.36 0.04 * . F 1.081.12
Thr 239 . . . . . . C 1.36 0.04 * . F 1.121.82
Leu 240 . . . . . . C 1.06 -0.17* . F 1.961.89
Pro 241 . . . . T . . 0.99 -0.21. . F 2.401.46
Asn 242 . . . . T . . 0.96 0.36 . . F 1.410.54
Asn 243 . . . . T T . 0.66 0.63 . . F 1.221.03
Ser 244 . . . . T T . 0.38 0.33 . . F 1.130.89
Cys 245 . . . . T T . 0.84 0.40 . . . 0.740.56
Tyr 246 . . . . T T . 0.17 0.43 . . . 0.200.35
Ser 247 A . . . . . . -0.420.71 . . . -0.400.18
Ala 248 A A . . . . . -0.380.83 . . . -0.600.34
Gly 249 A A . . . . . -0.890.26 . . . -0.300.43
Ile 250 A A . . . . . -0.220.19 * . . -0.300.27
CA 02467521 2004-05-14
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Table 9 (continued)
Res I II III IV V VI VII VIII IX X XI XII XIIIXIV
Posirion
Ala 251 A A . . . . . 0.02 -0.20* . . 0.300.46
Lys 252 A A . . . . . -0.02 -0.70. . . 0.600.80
Leu 253 A A . . . . . 0.57 -0.70. . F 0.901.13
Glu 254 A A . . . . . 0.91 -1.39. . F 0.901.87
Glu 255 A A . . . . . 0.99 -1.89. . F 0.901.62
Gly 256 A A . . . . . 1.58 -1.20. * F 0.901.62
Asp 257 A A . . . . . 0.72 -1.49. * F 0.901.62
Glu 258 A A . . . . . 0.94 -0.80* * F 0.750.77
Leu 259 A A . . . . . 0.06 -0.30* * . 0.300.79
Gln 260 A A . . . . . -0.16 -0.04* . . 0.300.33
Leu 261 A A . . . . . 0.30 0.39 * . . -0.300.30
Ala 262 A A . . . . . 0.30 0.39 * . . -0.300.70
Ile 263 A A . . . . . 0.30 -0.30. * . 0.300.70
Pro 264 A . . . . T . 0.52 -0.30. * F 1.001.37
Arg 265 A . . . . T . 0.52 -0.49. * F 1.001.37
Glu 266 A . . . . T . 0.44 -0.59* * F 1.303.38
Asn 267 A . . . . T . 0.73 -0.59* * F 1.301.53
Ala 268 A . . . . . . 0.81 -0.63* * . 0.951.05
Gln 269 A . . . . . . 1.02 0.06 * * . -0.100.50
Ile 270 A . . . . . . 0.57 0.06 . * . 0.150.52
Ser 271 . . . . . . C 0.57 0.09 . * . 0.600.51
Leu 272 . . . . . . C -0.29 -0.41. * F 1.600.49
Asp 273 . . . . T T . -0.01 -0.17. * F 2.250.52
Gly 274 . . . . T T . -0.71 -0.37. * F 2.500.56
Asp 275 . . . . T T . -0.52 0.03 . * F 1.650.59
Val 276 A . . . . T . -0.57 0.13 . * F 1.000.30
Thr 277 A . . B . . . -0.34 0.56 . * . -0.100.30
Phe 278 A . . B . . . -1.16 0.63 . * . -0.350.18
Phe 279 A . . B . . . -0.77 1.31 . * . -0.600.20
Gly 280 A A . . . . . -1.58 0.67 . * . -0.600.28
Ala 281 A A . . . . . -1.53 0.87 . * . -0.600.27
Leu 282 A A . . . . . -1.61 0.77 * . . -0.600.26
Lys 283 A A . . . . . -1.30 0.41 * . . -0.600.33
Leu 284 A A . . . . . -0.99 0.41 . . . -0.600.42
Leu 285 A A . . . . . -1.03 0.34 * . . -0.300.65
41
CA 02467521 2004-05-14
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Table 10
Res I II III IV V VI VII VIII IX X XI XII XIIIXN
Position
Met 1 A . . . . . . 0.73 -0.71. . . 0.951.39
Asp 2 A . . . . T . 1.12 -0.66* . . 1.151.56
Asp 3 A . . . . T . 1.62 -1.09* . . 1.152.12
Ser 4 A . . . . T . 2.01 -1.51. . . 1.154.19
Thr 5 A . . . . T . 2.40 -2.13. . F 1.304.35
Glu 6 A A . . . . . 2.70 -1.73* * F 0.904.51
Arg 7 A A . . . . . 2.81 -1.34* * F 0.904.51
Glu 8 A A . . . . . 2.00 -1.73* * F 0.906.12
Gln 9 A A . . . . . 1.99 -1.53* * F 0.902.91
Ser 10 A . . B . . . 2.00 -1.04* * F 0.902.15
Arg 11 A . . B . . . 1.33 -0.66* * F 0.901.66
Leu 12 A . . B . . . 0.41 -0.09* * F 0.450.51
Thr 13 A . . B . . . 0.46 0.20 * * F -0.150.32
Ser 14 A A . . . . . 0.50 -0.19* * . 0.300.32
Cys 15 A A . . . . . 0.91 -0.19* * . 0.300.78
Leu 16 A A . . . . . 0.80 -0.87* * F 0.901.06
Lys 17 A A . . . . . 1.61 -1.36. * F 0.901.37
Lys 18 A A . . . . . 1.32 -1.74. * F 0.904.44
Arg 19 A A . . . . . 1.67 -1.70. * F 0.905.33
Glu 20 A A . . . . . 1.52 -2.39. * F 0.905.33
Glu 21 A A . . . . . 2.38 -1.70. * F 0.902.20
Met 22 A A . . . . . 2.33 -1.70. * F 0.902.24
Lys 23 A A . . . . . 1.62 -1.70* * F 0.902.24
Leu 24 A A . . . . . 0.66 -1.13* * F 0.750.69
Lys 25 A A . . . . . 0.36 -0.49. * F 0.450.52
Glu 26 A A . B . . . -0.53-0.71* * . 0.600.35
Cys 27 A A . B . . . -0.74-0.03* * . 0.300.30
Val 28 A A . B . . . -1.00-0.03* * . 0.300.12
Ser 29 A A . B . . . -0.080.40 * * . -0.300.11
Ile 30 A . . B . . . -0.080.40 * * . -0.300.40
Leu 31 A . . B . . . -0.08-0.17* . . 0.451.08
Pro 32 . . . B . . C 0.29 -0.81* . F 1.101.39
Arg 33 . . . . T . . 0.93 -0.81. * F 1.502.66
Lys 34 . . . . T . . 0.93 -1.07. . F 1.844.98
Glu 35 . . . . . . C 0.97 -1.37* * F 1.984.32
- Ser 36 . . . . - T C 1.89 -1.16* - F 2.521.64
. - * --
-
Pro 37 . . . . . T C 1.80 -1.16* * F 2.861.60
Ser 38 . . . . T T . 1.39 -0.77* . F 3.401.24
Val 39 A . . . . T . 1.39 -0.39. * F 2.361.24
Arg 40 A . . . . . . 1.39 -0.77* * F 2.461.60
Ser 41 A . . . . . . 1.34 -1.20* * F 2.462.00
Ser 42 . . . . T T . 1.60 -1.16. * F 3.062.67
Lys 43 . . . . T T . 1.09 -1.80* * F 3.062.72
Asp 44 . . . . T T . 1.13 -1.11* * F 3.401.67
Gly 45 A . . . . T . 0.43 -0.81* * F 2.661.03
Lys 46 A A . . . . . 0.14 -0.70. . F 1.770.52
Leu 47 A A . . . . . 0.13 -0.20* . . 0.980.31
Leu 48 A A . . . . . -0.720.29 * . . 0.040.46
Ala 49 A A . . . . . -1.530.54 . * . -0.600.19
Ala 50 A A . . . . . -2.001.23 . . . -0.600.19
42
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
Table 10 (continued)
Res I II IiI N V VI VII VIII IX X XI XII XIIIXIV
Posirion
Thr 51 A A . . . . . -2.63 1.23 . . -0.600.19
.
Leu 52 A A . . . . . -2.63 1.04 . . -0.600.19
.
Leu 53 A A . . . . . -2.63 1.23 . . -0.600.15
.
Leu 54 A A . . . . . -2.34 1.41 . . -0.600.09
.
Ala 55 A A . . . . . -2.42 1.31 . . -0.600.14
.
Leu 56 A A . . . . . -2.78 1.20 . . -0.600.09
.
Leu 57 A . . . . T . -2.78 1.09 . . -0.200.06
.
Ser 58 A . . . . T . -2.28 1.09 . . -0.200.05
.
Cys 59 A . . . . T . -2.32 1.07 . . -0.200.09
.
Cys 60 A . . . . T . -2.59 1.03 . . -0.200.08
.
Leu 61 . . B B . . . -2.08 0.99 . . -0.600.04
.
Thr 62 . . B B . . . -1.97 0.99 . . -0.600.11
.
Val 63 . . B B . . . -1.91 1.20 . . -0.600.17
.
Val 64 . . B B . . . -1.24 1.39 . . -0.600.33
.
Ser 65 . . B B . . . -1.43 1.10 . . -0.600.40
.
Phe 66 A . . B . . . -1.21 1.26 . . -0.600.40
.
Tyr 67 A . . B . . . -1.49 1.11 . . -0.600.54
.
Gln 68 A . . B . . . -1.44 0.97 . . -0.600.41
.
Val 69 A . . B . . . -0.59 1.27 . . -0.600.39
.
Ala 70 A . . B . . . -0.63 0.89 . . -0.600.43
.
Ala 71 A . . B . . . 0.07 0.56 * . -0.600.25
.
Leu 72 A . . . . T . -0.50 0.16 . . 0.100.55
.
Gln 73 A . . . . T . -1.09 0.20 . F 0.250.45
.
Gly 74 A . . . . T . -0.53 0.20 . F 0.250.45
.
Asp 75 A . . . . T . -0.76 0.09 * F 0.250.73
.
Leu 76 A A . . . . . -0.06 0.09 * F -0.150.35
.
Ala 77 A A . . . . . 0.17 -0.31 * . 0.300.69
.
Ser 78 A A . . . . . 0.17 -0.24 * . 0.300.42
.
Leu 79 A A . . . . . -0.30 -0.24 * . 0.300.88
.
Arg 80 A A . . . . . -0.30 -0.24 * . 0.300.72
.
Ala 81 A A . . . . . 0.17 -0.34 * . 0.300.93
.
Glu 82 A A . . . . . 0.72 -0.30 * . 0.451.11
.
Leu 83 A A . . . . . 0.99 -0.49 * . 0.300.77
.
Gln 84 A A . . . . . 1.21 0.01 * . -0.151.04
.
Gly 85 A A . . . . . 1.10 0.01 * . -0.300.61
*
_ _ 86 A A - . - . __ _ _ 0.01 * . -0.151.27
_ His - . . __ 1.73 * _
_
His 87 A A . . . . . 0.92 -0.67 * . 0.751.47
.
Ala 88 A A . . . . . 1.52 -0.39 * . 0.451.22
.
Glu 89 A A . . . . . 0.93 -0.39 . . 0.451.39
.
Lys 90 A A . . . . . 0.93 -0.39 . F 0.601.03
*
Leu 91 A . . . . T . 0.38 -0.46 . . 0.851.01
*
Pro 92 A . . . . T . 0.07 -0.46 . . 0.700.59
.
Ala 93 A . . . . T . 0.07 -0.03 . . 0.700.29
.
Gly 94 A . . . . T . -0.14 0.47 . . -0.200.36
.
Ala 95 A . . . . . . -0.14 0.21 * . -0.100.36
.
Gly 96 A . . . . . . 0.08 -0.21 . F 0.650.71
.
Ala 97 A . . . . . . -0.06 -0.21 . F 0.650.72
.
Pro 98 A . . . . . . -0.28 -0.21 * F 0.650.71
.
Lys 99 A A . . . . . 0.07 -0.03 . F 0.450.59
.
Ala 100 A A . . . . . 0.66 -0.46 . F 0.601.01
.
43
CA 02467521 2004-05-14
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Table 10 (continued)
Res I II III IV V VI VII VIII IX X XI XII XIIIXN
Position
Gly 101 A A . . . . . 0.41 -0.96. . F 0.901.13
Leu 102 A A . . . . . 0.79 -0.89. . F 0.750.57
Glu 103 A A . . . . . 0.41 -0.46* . F 0.450.88
Glu 104 A A . . . . . -0.49-0.46* . F 0.450.89
Ala 105 A A . . . . . -0.21-0.24. . . 0.300.81
Pro 106 A A . . . . . -0.46-0.44. . . 0.300.67
Ala 107 A A . . . . . 0.01 0.06 . . . -0.300.39
Val 108 A A . . . . . -0.800.49 * * . -0.600.38
Thr 109 A A . . . . . -0.760.67 . * . -0.600.20
Ala 110 A A . . . . . -1.060.24 * * . -0.300.40
Gly 111 A A . . . . . -1.540.43 * * . -0.600.38
Leu 112 A A . . . . . -0.960.57 * * . -0.600.23
Lys 113 . A B . . . . -0.310.09 * * . -0.300.39
Ile 114 . A B . . . . -0.210.01 * . . -0.300.61
Phe 115 . A B . . . . -0.210.01 * . . 0.151.15
Glu 116 . A . . . . C -0.08-0.17* . F 1.250.58
Pro 117 . A . . . . C 0.39 0.26 * * F 1.101.28
Pro 118 . . . . . . C 0.34 0.00 * . F 2.201.47
Ala 119 . . . . . T C 0.89 -0.79. * F 3.001.47
Pro 120 . . . . . T C 1.59 -0.36. * F 2.250.94
Gly 121 . . . . T T . 1.29 -0.39. * F 2.150.98
Glu 122 . . . . T T . 1.20 -0.43. . F 2.001.30
Gly 123 . . . . . . C 1.41 -0.54. . F 1.601.12
Asn 124 . . . . . T C 2.00 -0.57. . F 1.501.97
Ser 125 . . . . . T C 1.91 -0.60. * F 1.501.82
Ser 126 . . . . . T C 2.37 -0.21. * F 1.542.47
Gln 127 . . . . . T C 2.37 -0.64. * F 2.183.01
Asn 128 . . . . . . C 2.76 -0.64. . F 2.323.61
Ser 129 . . . . . T C 2.87 -1.03. . F 2.865.39
Arg 130 . . . . T T . 2.58 -1.41* . F 3.406.09
Asn 131 . . . . T T . 2.02 -1.31* . F 3.063.83
Lys 132 . . . . T T . 2.02 -1.07* . F 2.722.12
Arg 133 . . . . T . . 1.68 -1.06* . F 2.181.88
Ala 134 . . . . . . C 1.77 -0.63* . F 1.641.15
Val 135 . . . . . . C 1.66 -0.60* . F 1.150.89
Gln 136 _ . . , , , C 1.66 _0.60*., F _ 0.79
.. . . -._ 1.49
_
Gly 137 . . . . . T C 1.30 -0.60* . F 2.181.35
Pro 138 . . . . . T C 0.84 -0.61* . F 2.522.63
Glu 139 . . . . . T C 1.13 -0.83* . F 2.861.50
Glu 140 . . . . T T . 1.74 -0.84. . F 3.402.03
Thr 141 . . . . T . . 1.43 -0.51. . F 2.862.06
Gly 142 . . . . T T . 1.08 -0.46. . F 2.421.72
Ser 143 . . . . T T . 0.43 0.33 . . F 1.330.86
Tyr 144 . . . . T T . 0.22 0.97 . . . 0.540.44
Thr 145 . . . . T T . -0.070.91 . . . 0.200.69
Phe 146 . . B B . . . -0.571.40 . . . -0.600.54
Val 147 . . B B . . . -1.031.70 . . . -0.600.29
Pro 148 . . B B . . . -1.031.63 . . . -0.600.16
Trp 149 A . . B . . . -1.491.53 . * . -0.600.25
Leu 150 A . . B . . . -1.131.53 * . . -0.600.29
44
CA 02467521 2004-05-14
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Table 10 (continued)
Res I II III IV V VI VII VIII IX X XI XII XIIIXIV
Position
Leu 151 A . . B . . . -0.320.89 * . . -0.300.38
Ser 152 A . . . . . . 0.19 0.46 * . . 0.200.71
Phe 153 . . . . T . . 0.10 -0.03* . . 1.800.85
Lys 154 . . . . T T . -0.20-0.33* . F 2.601.38
Arg 155 . . . . . T C -0.20-0.51. . F 3.001.04
Gly 156 . . . . . T C 0.61 -0.21. . F 2.250.99
Ser 157 A . . . . T . 0.91 -1.00* . F 2.050.86
Ala 158 A A . . . . . 1.66 -1.00* . F 1.350.76
Leu 159 A A . . . . . 1.61 -1.00. . F 1.201.54
Glu 160 A A . . . . . 1.50 -1.43. . F 0.901.98
Glu 161 A A . . . . . 1.89 -1.41* . F 0.903.16
Lys 162 A A . . . . . 1.30 -1.91* . F 0.907.66
Glu 163 A A . . . . . 1.08 -1.91. . F 0.903.10
Asn 164 A A . . . . . 1.03 -1.23* * F 0.901.48
Lys 165 A A . . . . . 1.08 -0.59* . F 0.750.55
Ile 166 A A . . . . . 1.08 -0.59* * . 0.600.63
Leu 167 A A . . . . . 0.72 -0.59* * . 0.760.68
Val 168 A A . . . . . 0.38 -0.50. * . 0.920.49
Lys 169 A A . . . . . 0.13 -0.07* * F 0.930.69
Glu 170 A . . . . T . -0.610.00 * * F 1.641.32
Thr 171 . . . . T T . -0.420.10 . * F 1.601.54
Gly 172 . . . . T T . -0.500.24 * . F 1.290.67
Tyr 173 . . . . T T . 0.11 0.93 * * . 0.680.27
Phe 174 . . B B . . . -0.281.69 . . . -0.280.29
Phe 175 . . B B . . . -0.281.63 . * . -0.440.29
Ile 176 . . B B . . . -0.821.60 . . . -0.600.32
'
Tyr 177 . . B B . . . -1.291.49 . . . -0.600.28
Gly 178 . . . B T . . -1.291.39 . . . -0.200.26
Gln 179 . . . B T . . -0.901.36 . . . -0.200.59
Val 180 . . . B . . C -0.201.16 . . . -0.400.54
Leu 181 . . . B . . C 0.73 0.40 . . . -0.100.92
Tyr 182 . . . . T T . 0.67 -0.03. . . 1.251.06
Thr 183 . . . . T T . 0.77 0.06 . . F 0.802.06
Asp 184 . . . . T T . 0.18 0.17 . . F 0.803.91
Lys 185 A . . . . T . 0.43 -0.01. . F 1.002.52
Thr-186 A A . , , , , 0.90 _0.16- . F- 0.60-
_ - . _- 1.73
Tyr 187 A A . . . . . 1.11 -0.21. . . 0.451.03
Ala 188 A A . . . . . 0.61 0.29 . . . -0.300.70
Met 189 A A . . . . . -0.280.97 . . . -0.600.40
Gly 190 A A . B . . . -0.321.17 * . . -0.600.18
His 191 A A . B . . . 0.10 0.81 * . . -0.600.31
Leu 192 A A . B . . . 0.39 0.31 . . . -0.300.61
Ile 193 A A . B . . . 1.02 -0.30. . . 0.451.22
Gln 194 A A . B . . . 0.77 -0.73. * . 0.751.80
Arg 195 A A . B . . . 1.08 -0.59* * F 0.901.62
Lys 196 A A . B . . . 0.26 -0.77* * F 0.903.14
Lys 197 A A . B . . . 0.37 -0.81. * F 0.901.35
Val 198 . A B B . . . 0.91 -0.43* * . 0.300.60
His 199 . A B B . . . 0.91 0.00 * * . 0.300.29
Val 200 . A B B . . . 0.80 0.00 * * . 0.300.25
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
Table 10 (continued)
Res I II III N V VI VIIVIII IX X XI XII XIIIXIV
Position
Phe 201 . . B B . . . -0.060.00 * . . 0.300.57
Gly 202 A . . B . . . -0.400.04 . * . -0.300.35
Asp 203 A . . . . . . -0.36-0.07* . . 0.500.63
Glu 204 A . . . . . . -1.18-0.03* . . 0.500.60
Leu 205 A . . B . . . -0.63-0.17. . . 0.300.45
Ser 206 A . . B . . . -0.74-0.11. . . 0.300.39
Leu 207 A . . B . . . -1.100.57 . * . -0.600.18
Val 208 A . . B . . . -0.991.36 . * . -0.600.19
Thr 209 A . . B . . . -1.660.67 * * . -0.600.28
Leu 210 A . . B . . . -1.730.86 * . . -0.600.18
Phe 211 A . . B . . . -1.430.86 * . . -0.600.17
Arg 212 A . . B . . . -0.620.61 * . . -0.600.21
Cys 213 . . . B T . . -0.370.53 * . . -0.200.41
Ile 214 . . . B T . . -0.270.46 * . . -0.200.46
Gln 215 . . . B T . . 0.54 0.10 * . . 0.100.37
Asn 216 . . . B . . C 0.93 0.10 * . . 0.051.19
Met 217 . . . B . . C 0.01 0.01 * . F 0.202.44
Pro 218 . . . B . . C 0.47 0.01 * . F 0.441.16
Glu 219 . . . . T . . 1.36 0.04 * . F 1.081.12
Thr 220 . . . . . . C 1.36 0.04 * . F 1.121.82
Leu 221 . . . . . . C 1.06 -0.17* . F 1.961.89
Pro 222 . . . . T . . 0.99 -0.21. . F 2.401.46
Asn 223 . . . . T . . 0.96 0.36 . . F 1.410.54
Asn 224 . . . . T T . 0.66 0.63 . . F 1.221.03
Ser 225 . . . . T T . 0.38 0.33 . . F 1.130.89
Cys 226 . . . . T T . 0.84 0.40 . . . 0.740.56
Tyr 227 . . . . T T . 0.17 0.43 . . . 0.200.35
Ser 228 A . . . . . . -0.420.71 . . . -0.400.18
Ala 229 A A . . . . . -0.380.83 . . . -0.600.34
Gly 230 A A . . . . . -0.890.26 . . . -0.300.43
Ile 231 A A . . . . . -0.220.19 * . . -0.300.27
Ala 232 A A . . . . . 0.02 -0.20* . . 0.300.46
Lys 233 A A . . . . . -0.02-0.70. . . 0.600.80
Leu 234 A A . . . . . 0.57 -0.70. . F 0.901.13
Glu 235 A A . . . . . 0.91 -1.39. . F 0.901.87
Glu 236 - A . . . . . 0:99 -1.89. . F. - 1.62
A - 0.90-
Gly 237 A A . . . . . 1.58 -1.20. * F 0.901.62
Asp 238 A A . . . . . 0.72 -1.49. * F 0.901.62
Glu 239 A A . . . . . 0.94 -0.80* * F 0.750.77
Leu 240 A A . . . . . 0.06 -0.30* * . 0.300.79
Gln 241 A A . . . . . -0.16-0.04* . . 0.300.33
Leu 242 A A . . . . . 0.30 0.39 * . . -0.30030
Ala 243 A A . . . . . 0.30 0.39 * . . -0.300.70
Ile 244 A A . . . . . 0.30 -0.30. * . 0.300.70
Pro 245 A . . . . T . 0.52 -0.30. * F 1.001.37
Arg 246 A . . . . T . 0.52 -0.49. * F 1.001.37
Glu 247 A . . . . T . 0.44 -0.59* * F 1.303.38
Asn 248 A . . . . T . 0.73 -0.59* * F 1.301.53
Ala 249 A . . . . . . 0.81 -0.63* * . 0.951.05
Gln 250 A . . . . . . 1.02 0.06 * * . -0.100.50
46
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Table 10 (continued)
Res I II III IV V VI VII VIII IX X XI XII XIIIXIV
Position
Ile 251 A . . . . . . 0.57 0.06 * . 0.150.52
*
Ser 252 . . . . . . C 0.57 0.09 * . 0.600.51
.
Leu 253 . . . . . . C -0.29 -0.41 * F 1.600.49
.
Asp 254 . . . . T T . -0.01 -0.17 * F 2.250.52
.
Gly 255 . . . . T T . -0.71 -0.37 * F 2.500.56
.
Asp 256 . . . . T T . -0.52 0.03 * F 1.650.59
.
Val 257 A . . . . T . -0.57 0.13 * F 1.000.30
.
Thr 258 A . . B . . . -0.34 0.56 * . -0.100.30
.
Phe 259 A . . B . . . -1.16 0.63 * . -0.350.18
.
Phe 260 A . . B . . . -0.77 1.31 * . -0.600.20
.
Gly 261 A A . . . . . -1.58 0.67 * . -0.600.28
.
Ala 262 A A . . . . . -1.53 0.87 * . -0.600.27
.
Leu 263 A A . . . . . -1.61 0.77 . . -0.600.26
*
Lys 264 A A . . . . . -1.30 0.41 . . -0.600.33
*
Leu 265 A A . . . . . -0.99 0.41 . . -0.600.42
.
Leu 266 A A . . . . . -1.03 0.34 . . -0.300.65
*
47
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[0093] In another embodiment, the invention provides antibodies that bind a
polypeptide comprising, or alternatively consisting of, an epitope-bearing
portion of a
polypeptide of the invention. The epitope of this polypeptide portion may be
an
immunogenic or antigenic epitope of a polypeptide of the invention. An
"immunogenic
epitope" is defined as a part of a protein that elicits an antibody response
when the whole
protein is the immunogen. On the other hand, a region of a protein molecule to
which an
antibody can bind is defined as an "antigenic epitope." The number of
immunogenic
epitopes of a protein generally is less than the number of antigenic epitopes.
See, for
instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983).
[0094] As to the selection of polypeptides bearing an antigenic epitope (i.e.,
that
contain a region of a protein molecule to which an antibody can bind), it is
well known in
that art that relatively short synthetic peptides that mimic part of a protein
sequence are
routinely capable of eliciting an antiserum that reacts with the partially
mimicked protein.
See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner,
R. A. (1983)
"Antibodies that react with predetermined sites on proteins", Scieyzce,
219:660-666.
Peptides capable of eliciting protein-reactive sera are frequently represented
in the primary
sequence of a protein, can be characterized by a set of simple chemical rules,
and are
confined neither to immunodominant regions of intact proteins (i.e.,
immunogenic
epitopes) nor to the amino or carboxyl terminals. Antigenic epitope-bearing
peptides and
polypeptides of the invention are therefore useful to raise antibodies,
including
monoclonal antibodies, that bind specifically to a polypeptide of the
invention. See, for
instance, Wilson et al., Cell 37:767-778 (1984) at 777.
[0095] In specific embodiments, antibodies of the present invention bind
antigenic
epitope-bearing peptides and polypeptides of BLyS and preferably contain a
sequence of
at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at
least 25, at least 30, at
least 40, at least 50, and, most preferably, between about 15 to about 30
amino acids
contained within the amino acid sequence of a BLyS polypeptide. Preferred
polypeptides
comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30,
35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
Additional
non-exclusive preferred antigenic epitopes include the antigenic epitopes
disclosed herein,
as well as portions thereof.
48
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[0096] Non-limiting examples of antigenic polypeptides or peptides that can be
used
to generate BLyS-specific antibodies and which may be bound by the antibodies
of the
invention include: a polypeptide comprising, or alternatively consisting of,
amino acid
residues from about Phe-115 to about Leu-147 in SEQ ID N0:3228; a polypeptide
comprising, or alternatively consisting of, amino acid residues from about Ile-
150 to
about Tyr-163 in SEQ ID N0:3228; a polypeptide comprising, or alternatively
consisting
of, amino acid residues from about Ser-171 to about Phe-194 in SEQ ID NO:3228;
a
polypeptide comprising, or alternatively consisting of, amino acid residues
from about
Glu-223 to about Tyr-246 in SEQ ID N0:3228; and a polypeptide comprising, or
alternatively consisting of, amino acid residues from about Ser-271 to about
Phe-278 in
Figures 1A and 1B (SEQ ID N0:3228). In this context, "about" means the
particularly
recited ranges and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1
amino acid
residues at either or both the amino- and carboxy-termini. These polypeptide
fragments
have been determined to bear antigenic epitopes of the BLyS polypeptide by the
analysis
of the Jameson-Wolf antigenic index, as disclosed Table 9, above.
[0097] Non-limiting examples of antigenic polypeptides or peptides that can be
used
to generate BLyS-specific antibodies and which may be bound by the antibodies
of the
invention include: a polypeptide comprising, or alternatively consisting of,
amino acid
residues from about Pro-32 to about Leu-47 in SEQ 117 N0:3229; a polypeptide
comprising, or alternatively consisting of, amino acid residues from about Glu-
116 to
about Ser-143 in SEQ ID NO:3229; a polypeptide comprising, or alternatively
consisting
of, amino acid residues from about Phe-153 to about Tyr-173 in SEQ ID N0:3229;
a
polypeptide comprising, or alternatively consisting of, amino acid residues
from about
Pro-218 to about Tyr-227 in SEQ ID N0:3229; a polypeptide comprising, or
alternatively
consisting of, amino acid residues from about Ala-232 to about Gln-241 in SEQ
ID
N0:3229; a polypeptide comprising, or alternatively consisting of, amino acid
residues
from about Ile-244 to about Ala-249 in SEQ ID N0:3229; and a polypeptide
comprising,
or alternatively consisting of, amino acid residues from about Ser-252 to
about Val-257 in
SEQ ID N0:3229. In this context, "about" means the particularly recited ranges
and
ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid
residues at either or
both the amino- and carboxy-termini. These polypeptide fragments have been
determined
to bear antigenic epitopes of the BLyS polypeptide by the analysis of the
Jameson-Wolf
49
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WO 03/055979 PCT/US02/36496
antigenic index, as disclosed in Table 10 generated by the Protean component
of the
DNA*STAR computer program (as set forth above).
[0098] BLyS epitope-bearing peptides and polypeptides may be produced by any
conventional means. See, e.g., Houghten, R. A. (1985) General method for the
rapid
solid-phase synthesis of large numbers of peptides: specificity of antigen-
antibody
interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA
82:5131-5135; this "Simultaneous Multiple Peptide Synthesis (SMPS)" process is
further
described in U. S. Patent No. 4,631,211 to Houghten et al. (1986).
[0099] The present invention encompasses antibodies that bind polypeptides
comprising, or alternatively consisting of, an epitope of the polypeptide
having an amino
acid sequence of SEQ m N0:3228, or an epitope of the polypeptide sequence
encoded by
a polynucleotide sequence contained in ATCC deposit No. 97768, or encoded by a
polynucleotide that hybridizes to cDNA sequence contained in ATCC deposit No.
97768
(e.g., under hybridization conditions described herein).
[0100] The present invention also encompasses antibodies that bind
polypeptides
comprising, or alternatively consisting of, an epitope of the polypeptide
having an amino
acid sequence of SEQ )D N0:3229, or an epitope of the polypeptide sequence
encoded by
a polynucleotide sequence contained in ATCC deposit No. 203518, or encoded by
a
polynucleotide that hybridizes to the cDNA sequence contained in ATCC deposit
No.
203518 (e.g., under hybridization conditions described herein).
[0101] The term "epitopes," as used herein, refers to portions of a
polypeptide having
antigenic or immunogenic activity in an animal, preferably a mammal, and most
preferably in a human. In a preferred embodiment, the present invention
encompasses
antibodies that bind a polypeptide comprising an epitope. An "immunogenic
epitope," as
used herein, is defined as a portion of a protein that elicits an antibody
response in an
animal, as determined by any method known in the art, for example, by the
methods for
generating antibodies described infra. (See, for example, Geysen et al., Proc.
Natl. Acad.
Sci. USA 81:3998- 4002 (1983)). The term "antigenic epitope," as used herein,
is defined
as a portion of a protein to which an antibody can immunospecifically bind its
antigen as
determined by any method well known in the art, for example, by the
immunoassays
described herein. Immunospecific binding excludes non-specific binding but
does not
CA 02467521 2004-05-14
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necessarily exclude cross- reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
[0102] BLyS polypeptide fragments which function as epitopes may be produced
by
any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA
82:5131-5135
(1985), further described in U.S. Patent No. 4,631,211).
[0103] In the present invention, antibodies of the present invention bind
antigenic
epitopes preferably containing a sequence of at least 4, at least 5, at least
6, at least 7, more
preferably at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at
least 15, at least 20, at least 25, at least 30, at least 40, at least 50,
and, most preferably,
between about 15 to about 30 amino acids. Preferred polypeptides comprising
immunogenic or antigenic epitopes that may be bound by antibodies of the
present
invention are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, or
100 amino acid residues in length. Additional non-exclusive preferred
antigenic epitopes
include the antigenic epitopes disclosed herein, as well as portions thereof.
Antigenic
epitopes are useful, for example, to raise antibodies, including monoclonal
antibodies, that
specifically bind the epitope. Preferred antigenic epitopes include the
antigenic epitopes
disclosed herein, as well as any combination of two, three, four, five or more
of these
antigenic epitopes. Antigenic epitopes can be used as the target molecules in
immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984);
Sutcliffe et al.,
Science 219:660-666 (1983)).
[0104] Similarly, immunogenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe et al.,
supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-
914; and
Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic
epitopes include
the immunogenic epitopes disclosed herein, as well as any combination of two,
three, four,
five or more of these immunogenic epitopes. The polypeptides comprising one or
more
immunogenic epitopes of BLyS may be presented for eliciting an antibody
response
together with a carrier protein, such as an albumin, to an animal system (such
as rabbit or
mouse), or, if the polypeptide is of sufficient length (at least about 25
amino acids), the
polypeptide may be presented without a carrier. However, immunogenic epitopes
comprising as few as 8 to 10 amino acids have been shown to be sufficient to
raise
51
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WO 03/055979 PCT/US02/36496
antibodies capable of binding to, at the very least, linear epitopes in a
denatured
polypeptide (e.g., in Western blotting).
[0105] Epitope-bearing BLyS polypeptides may be used to induce antibodies
according to methods well known in the art including, but not limited to, in
vivo
immunization, in vitro immunization, and phage display methods. See, e.g.,
Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-
2354 (1985). If in
vivo immunization is used, animals may be immunized with free peptide;
however, anti-
peptide antibody titer may be boosted by coupling the peptide to a
macromolecular carrier,
such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For instance,
peptides
containing cysteine residues may be coupled to a Garner using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be
coupled to carriers using a more general linking agent such as glutaraldehyde.
Animals
such as rabbits, rats and mice are immunized with either free or carrier-
coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of emulsions
containing about
100 micrograms of peptide or carrier protein and Freund's adjuvant or any
other adjuvant
known for stimulating an immune response. Several booster injections may be
needed, for
instance, at intervals of about two weeks, to provide a useful titer of anti-
peptide antibody
which can be detected, for example, by ELISA assay using free peptide adsorbed
to a solid
surface. The titer of anti-peptide antibodies in serum from an immunized
animal may be
increased by selection of anti-peptide antibodies, for instance, by adsorption
to the peptide
on a solid support and elution of the selected antibodies according to methods
well known
in the art.
[0106] As one of skill in the art will appreciate, and as discussed above, the
antibodies
of the present invention may bind polypeptides comprising an immunogenic or
antigenic
epitope fused to other polypeptide sequences. For example, the BLyS
polypeptides may
be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or
portions
thereof (CHl, CH2, CH3, or any combination thereof and portions thereof), or
albumin
(including but not limited to recombinant human albumin or fragments or
variants thereof
(see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413
622, and U.S.
Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference
in their
entirety)), resulting in chimeric polypeptides. ' Such fusion proteins may
facilitate
purification and may increase half-life ita vivo. This has been shown for
chimeric proteins
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WO 03/055979 PCT/US02/36496
consisting of the first two domains of the human CD4-polypeptide and various
domains of
the constant regions of the heavy or light chains of marmnalian
immunoglobulins. See,
e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced
delivery of an
antigen across the epithelial barrier to the immune system has been
demonstrated for
antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or
Fc fragments
(see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins
that
have a disulfide-linked dimeric structure due to the IgG portion disulfide
bonds have also
been found to be more efficient in binding and neutralizing other molecules
than
monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J.
Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can
also be
recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin
("HA") tag or
flag tag) to aid in detection and purification of the expressed polypeptide.
For example, a
system described by Janknecht et al. allows for the ready purification of non-
denatured
fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc.
Natl. Acad.
Sci. USA 88:8972- 897). In this system, the gene of interest is subcloned into
a vaccinia
recombination plasmid such that the open reading frame of the gene is
translationally
fused to an amino-terminal tag consisting of six histidine residues. The tag
serves as a
matrix-binding domain for the fusion protein. Extracts from cells infected
with the
recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose
column and
histidine-tagged proteins can be selectively eluted with imidazole-containing
buffers.
[0107] In another embodiment, the antibodies of the presentinvention bind BLyS
polypeptides and/or the epitope-bearing fragments thereof that are fused with
a
heterologous antigen (e.g., polypeptide, carbohydrate, phospholipid, or
nucleic acid). In
specific embodiments, the heterologous antigen is an immunogen.
[0108] In a more specific embodiment, the heterologous antigen is the gp120
protein
of HIV, or a fragment thereof.
[0109] In another embodiment, antibodies of the present invention bind BLyS
polypeptides and/or the epitope-bearing fragments thereof that are fused with
polypeptide
sequences of another TNF ligand family member (or biologically active
fragments or
variants thereof). In a specific embodiment, the antibodies of the present
invention bind
BLyS polypeptides of the present invention are fused with a CD40L polypeptide
sequence. In a preferred embodiment, the CD40L polypeptide sequence is
soluble.
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[0110] In another embodiment, antibodies of the present invention bind mutant
BLyS
polypeptides that have been generated by random mutagenesis of a
polynucleotide
encoding the BLyS polypeptide, by error-prone PCR~ random nucleotide insertion
or other
methods prior to recombination. In another embodiment, antibodies of the
present
invention bind one or more components, motifs, sections, parts, domains,
fragments, etc.,
of BLyS recombined with one or more components, motifs, sections, parts,
domains,
fragments, etc. of one or more heterologous molecules. In preferred
embodiments, the
heterologous molecules are, for example, TNF-alpha, lymphotoxin-alpha (LT-
alpha, also
known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta),
OPGL,
Fast, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International
Publication No. WO 96/14328), AIM-I (International Publication No. WO
97/33899),
AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med.
188(6):1185-
1190), endokine-alpha (International Publication No. WO 98/07880), OPG, OX40,
and
nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-
IBB,
TR2 (International Publication No. WO 96/34095), DR3 (International
Publication No.
WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5
(International
Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694),
TR7
(International Publication No. WO 98/41629), TRANI~, TR9 (International
Publication
No. WO 98/56892), TR10 (International Publication No. WO 98/54202),312C2
(International Publication No. WO 98106842), TR12, CAD, and v-FLIP. In further
embodiments, the heterologous molecules are any member of the TNF family.
[0111] In another preferred embodiment, antibodies of the present invention
bind
BLyS polypeptides of the invention (including biologically active fragments or
variants
thereof), that are fused with soluble APRIL polypeptides (e.g., amino acid
residues 105
through 250 of SEQ ID N0:3239), or biologically active fragments or variants
thereof.
[0112] To improve or alter the characteristics of BLyS polypeptides, protein
engineering may be employed. Recombinant DNA technology known to those skilled
in
the art can be used to create novel mutant proteins or "muteins including
single or multiple
amino acid substitutions, deletions, additions or fusion proteins. Such
modified
polypeptides can show, e.g., enhanced activity or increased stability. In
addition, they
may be purified in higher yields and show better solubility than the
corresponding natural
polypeptide, at,least under certain purification and storage conditions. For
instance, for
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many proteins, including the extracellular domain or the mature forms) of a
secreted
protein, it is known in the art that one or more amino acids may be deleted
from the
N-terminus or C-terminus without substantial loss of biological function. For
instance,
Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF
proteins that had
heparin binding activity even if 3, 8, or 27 amino-terminal amino acid
residues were
missing. Accordingly, antibodies of the present invention may bind BLyS
polypeptide
mutants or variants generated by protein engineering.
[0113] In the present case, since the protein of the invention is a member of
the TNF
polypeptide family, deletions of N-terminal amino acids up to the Gly (G)
residue at
position 191 in SEQ ID N0:3228 may retain some biological activity such as,
for
example, the ability to stimulate lymphocyte (e.g., B cell) proliferation,
differentiation,
and/or activation, and cytotoxicity to appropriate target cells. Polypeptides
having further
N-terminal deletions including the Gly (G) residue would not be expected to
retain
biological activities because it is known that this residue in TNF-related
polypeptides is in
the beginning of the conserved domain required for biological activities.
However, even if
deletion of one or more amino acids from the N-terminus of a protein results
in
modification or loss of one or more biological functions of the protein, other
functional
activities may still be retained. Thus, the ability of the shortened protein
to induce and/or
bind to antibodies which recognize the complete or extracellular domain of the
protein
generally will be retained when less than the majority of the residues of the
complete or
extracellular domain of the protein are removed from the N-terminus. Whether a
particular polypeptide lacking N-terminal residues of a complete protein
retains such
immunologic activities can readily be determined by routine methods described
herein and
otherwise known in the art.
[0114] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the amino
acid sequence of the BLyS of SEQ ID NO:3228, up to the glycine residue at
position 191
(Gly-191 residue from the amino terminus). In particular, the present
invention provides
antibodies that bind polypeptides comprising, or alternatively consisting of,
the amino acid
sequence of residues n1-285 of SEQ ID N0:3228, where nl is an integer in the
range of the
amino acid position of amino acid residues 2-190 of the amino acid sequence in
SEQ ID
N0:3228. More in particular, the invention provides antibodies that bind
polypeptides
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
comprising, or alternatively consisting of, an amino acid sequence selected
from the group
consisting of residues 2-285, 3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285,
10-285,
11-285, 12-285, 13-285, 14-285, 15-285, 16-285, 17-285, 18-285, 19-285, 20-
285,
21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-
285,
31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-
285,
41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-
285,
51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, - 59-285, 60-
285,
61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-
285,
71-285, 72-285, 73-285, 74-285, 75-285, 76-285, 77-285, 78-285, 79-285, 80-
285,
81-285, 82-285, 83-285, 84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-
285,
91-285, 92-285, 93-285, 94-285, 95-285, 96-285, 97-285, 98-285, 99-285, 100-
285,
101-285, 102-285, 103-285, 104-285, 105-285, 106-285, 107-285, 108-285, 109-
285,
110-285, 111-285, 112-285, 113-285, 114-285, 115-285, 116-285, 117-285, 118-
285,
119-285, 120-285, 121-285, 122-285, 123-285, 124-285, 125-285, 126-285, 127-
285,
128-285, 129-285, 130-285, 131-285, 132-285, 133-285, 134-285, 135-285, 136-
285,
137-285, 138-285, 139-285, 140-285, 141-285, 142-285, 143-285, 144-285, 145-
285,
146-285, 147-285, 148-285, 149-285, 150-285, 151-285, 152-285, 153-285, 154-
285,
155-285, 156-285, 157-285, 158-285, 159-285, 160-285, 161-285, 162-285, 163-
285,
164-285, 165-285, 166-285, 167-285, 168-285, 169-285, 170-285, 171-285, 172-
285,
173-285, 174-285, 175-285, 176-285, 177-285, 178-285, 179-285, 180-285, 181-
285,
182-285, 183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285, and
190-285
of SEQ ID N0:3228. The present invention is also directed to antibodies that
bind BLyS
polypeptides comprising, or alternatively, consisting of, a contiguous
sequence of amino
acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical
to the
amino acid sequence of BLyS polypeptides described above.
[0115] Furthermore, since the predicted extracellular domain of the BLyS
polypeptides of the invention may itself elicit biological activity, deletions
of N- and
C-terminal amino acid residues from the predicted extracellular region of the
polypeptide
(spanning positions Gln-73 to Leu-285 of SEQ m N0:3228) may retain some
biological
activity such as, for example, ligand binding, stimulation of lymphocyte
(e.g., B cell)
proliferation, differentiation, and/or activation, and modulation of cell
replication or
modulation of target cell activities. However, even if deletion of one or more
amino acids
56
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WO 03/055979 PCT/US02/36496
from the N-terminus of the predicted extracellular domain of a BLyS
polypeptide results
in modification or loss of one or more biological functions of the
polypeptide, other
functional activities may still be retained. Thus, the ability of the
shortened polypeptides
to induce and/or bind to antibodies which recognize the complete or mature or
extracellular domains of the polypeptides generally will be retained when less
than the
majority of the residues of the complete or mature or extracellular domains of
the
polypeptides are removed from the N-terminus. Whether a particular polypeptide
lacking
N-terminal residues of a complete polypeptide retains such immunologic
activities can
readily be determined by routine methods described herein and otherwise known
in the art.
[0116] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the amino
acid sequence of BLyS shown in SEQ ID N0:3228, up to the glycine residue at
position
number 280. In particular, the present invention provides antibodies that bind
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of
residues n2-285 of SEQ ID N0:3228, where n2 is an integer in the range of the
amino acid
position of amino acid residues 73-280 in SEQ ID N0:3228, and 73 is the
position of the
first residue from the N-terminus of the predicted extracellular domain of the
BLyS
polypeptide (disclosed in SEQ ID N0:3228). More in particular, in certain
embodiments,
the invention provides antibodies that bind polypeptides comprising, or
alternatively
consisting of, an amino acid sequence selected from the group consisting of
residues of
Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-
78 to
L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-
285; Q-84
to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to
L-285;
K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-
95 to
L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-
285;
G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-
285; P-106
to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-
111 to
L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116
to L-285;
P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-
285; E-122
to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-
127 to
L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132
to
L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137
to
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L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142
to L-285;
T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-
285; Q-148
to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-
153 to
L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158
to L-285;
Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-
285; T-164
to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; 'L-
169 to
L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174
to L-285;
G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-
285; E-180
to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-
185 to
L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190
to
L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195
to L-285;
Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-
285;
Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-
285; Y-206
to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-
211 to
L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216
to L-285;
V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-
285;
D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-
285; V-227
to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-
232 to
L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237
to L-285;
E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-
285; N-243
to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-
248 to
L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253
to L-285;
E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-
285; L-259
to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-
264 to
L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269
to
L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274
to L-285;
D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-
285; and
G-280 to L-285 of SEQ ID N0:3228. The present invention is also directed to
antibodies
that bind BLyS polypeptides comprising, or alternatively, consisting of, a
contiguous
sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or
99% identical to the amino acid sequence of BLyS polypeptides described above.
Ss
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[0117] Highly preferred embodiments of the invention are directed to
antibodies that
bind polypeptides comprising, or alternatively consisting of, a polypeptide
having an
amino acid sequence least 80%, 85%, 90% identical and more preferably at least
95%,
96%, 97%, 98%, 99% or 100% identical to BLyS polypeptide having the amino acid
sequence at positions 134-285 of SEQ ID N0:3228.
[0118] Preferred embodiments of the invention are directed to antibodies that
bind
polypeptides comprising, or alternatively consisting of, a polypeptide having
an amino
acid sequence at least 90% identical to a BLyS polypeptide having the amino
acid
sequence at positions 134-285 of SEQ ID N0:3228. More preferred embodiments of
the
invention are directed to antibodies that bind polypeptides comprising, or
alternatively
consisting of, a polypeptide having an amino acid sequence at least 95%
identical to a
BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ
ll~
NO:3228. More preferred embodiments of the invention are directed to
antibodies that
bind polypeptides comprising, or alternatively consisting of, a polypeptide
having an
amino acid sequence at least 96% identical to a BLyS polypeptide having the
amino acid
sequence at positions 134-285 of SEQ ID NO:3228.
[0119] Additionally, more preferred embodiments of the invention are directed
to
antibodies that bind polypeptides comprising, or alternatively consisting of,
a polypeptide
having an amino acid sequence at least 97% to a BLyS polypeptide having the
amino acid
sequence at positions 134-285 of SEQ ID N0:3228. Additionally, more preferred
embodiments of the invention are directed to antibodies that bind polypeptides
comprising, or alternatively consisting of, a polypeptide having an amino acid
sequence at
least 98% to a BLyS polypeptide having the amino acid sequence at positions
134-285 of
SEQ ID N0:3228. Additionally, more preferred embodiments of the invention are
directed to antibodies that bind polypeptides comprising, or alternatively
consisting of, a
polypeptide having an amino acid sequence at least 99% identical to BLyS
polypeptide
having the amino acid sequence at positions 134-285 of SEQ ID N0:3228.
[0120] In specific embodiments, antibodies of the present invention bind
polypeptides
comprising, or alternatively consisting of, one of the following N-terminally
deleted
polypeptide fragments of BLyS: amino acid residues Ala-71 through Leu-285,
amino acid
residues Ala-81 through Leu-285, amino acid residues Leu-112 through Leu-285,
amino
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acid residues Ala-134 through Leu-285, amino acid residues Leu-147 through Leu-
285,
and amino acid residues Gly-161 through Leu-285 of SEQ ID N0:3228.
[0121] Similarly, many examples of biologically functional C-terminal deletion
polypeptides are known. For instance, Interferon gamma shows up to ten times
higher
activities by deleting 8-10 amino acid residues from the carboxy terminus of
the protein
(Dobeli et al., J. Biotechfaology 7:199-216 (1988). Since the present protein
is a member
of the TNF polypeptide family, deletions of C-terminal amino acids up to the
leucine
residue at position 284 are expected to retain most if not all biological
activity such as, for
example, ligand binding, the ability to stimulate lymphocyte (e.g., B cell)
proliferation,
differentiation, and/or activation, and modulation of cell replication.
Polypeptides having
deletions of up to about 10 additional C-terminal residues (i.e., up to the
glycine residue at
position 274) also may retain some activity such as receptor binding, although
such
polypeptides would lack a portion of the conserved TNF domain which extends to
about
Leu-284 of SEQ ID N0:3228. However, even if deletion of one or more amino
acids
from the C-terminus of a protein results in modification or loss of one or
more biological
functions of the protein, other functional activities may still be retained.
Thus, the ability
of the shortened protein to induce and/or bind to antibodies which recognize
the complete
or mature protein generally will be retained when less than the majority of
the residues of
the complete or mature protein are removed from the C-terminus. Whether a
particular
polypeptide lacking C-terminal residues of a complete protein retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise
known in the art.
[0122] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the carboxy terminus of
the amino
acid sequence of the BLyS polypeptide of SEQ ID NO:3228, up to the glycine
residue at
position 274 (Gly-274). In particular, the present invention provides
antibodies that bind
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of
residues 1-ml of the amino acid sequence in SEQ ID N0:3228, where ml is any
integer in
the range of the amino acid position of amino acid residues 274-284 in SEQ ID
N0:3228.
More in particular, the invention provides antibodies that bind BLyS
polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the group
consisting of residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281,
1-282,
CA 02467521 2004-05-14
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1-283 and 1-284 of SEQ m N0:3228. The present invention is also directed to
antibodies
that bind BLyS polypeptides comprising, or alternatively, consisting of, a
contiguous
sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or
99% identical to the amino acid sequence of BLyS polypeptides described above.
[0123] Also provided are antibodies that bind BLyS polypeptides comprising, or
alternatively consisting of, BLyS polypeptides with one or more amino acids
deleted from
both the amino and the carboxyl termini, which may be described generally as
having
residues nl-ml of SEQ m N0:3228, where nl and ml are integers as defined
above. Also
included are antibodies that bind a polypeptide comprising, or alternatively
consisting of, a
portion of the complete BLyS amino acid sequence encoded by the deposited cDNA
clone
contained in ATCC Accession No. 97768 where this portion excludes from 1 to
190
amino acids from the amino terminus or from 1 to 11 amino acids from the C-
terminus of
the complete amino acid sequence (or any combination of these N-terminal and C-
terminal
deletions) encoded by the cDNA clone in the deposited plasmid.
[0124] Similarly, deletions of C-terminal amino acid residues of the predicted
extracellular domain of BLyS up to the leucine residue at position 79 of SEQ m
N0:3228
may retain some biological activity, such as, for example, ligand binding,
stimulation of
lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation,
and modulation
of cell replication or modulation of target cell activities. Polypeptides
having further
C-terminal deletions including Leu-79 of SEQ ID N0:3228 would not be expected
to
retain biological activities.
[0125] However, even if deletion of one or more amino acids from the C-
terminus of a
polypeptide results in modification or loss of one or more biological
functions of the
polypeptide, other functional activities may still be retained. Thus, the
ability of the
shortened polypeptide to induce and/or bind to antibodies which recognize the
complete,
mature or extracellular forms of the polypeptide generally will be retained
when less than
the majority of the residues of the complete, mature or extracellular forms of
the
polypeptide are removed from the C-terminus. Whether a particular polypeptide
lacking
C-terminal residues of the predicted extracellular domain retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise
known in the art.
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[0126] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the carboxy terminus of
the amino
acid sequence of the predicted extracellular domain of BLyS polypeptide shown
in SEQ
ID NO:3228, up to the leucine residue at position 79 of SEQ ID N0:3228. In
particular,
the present invention provides antibodies that bind polypeptides comprising,
or
alternatively consisting of, the amino acid sequence of residues 73-m2 of the
amino acid
sequence in SEQ ID N0:3228, where m2 is any integer in the range of the amino
acid
position of amino acid residues 79 to 285 in the amino acid sequence in SEQ ID
N0:3228,
and residue 78 is the position of the first residue at the C- terminus of the
predicted
extracellular domain of the BLyS polypeptide (disclosed in SEQ ID N0:3228).
More in
particular, in certain embodiments, the invention provides antibodies that
bind
polypeptides comprising, or alternatively consisting of, an amino acid
sequence selected
from the group consisting of residues Q-73 to Leu-285; Q-73 to L-284; Q-73 to
K-283;
Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F-278; Q-
73 to
T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-
272;
Q-73 to S-271; Q-73 to I-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-
73 to
E-266; Q-73 to R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-
261; Q-73
to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to G-256; Q-73 to
E-255;
Q-73 to E-254; Q-73 to L-253; Q-73 to K-252; Q-73 to A-251; Q-73 to I-250; Q-
73 to
G-249; Q-73 to A-248; Q-73 to S-247; Q-73 to Y-246; Q-73 to C-245; Q-73 to S-
244;
Q-73 to N-243; Q-73 to N-242; Q-73 to P-241; Q-73 to L-240; Q-73 to T-239; Q-
73 to
E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234; Q-73 to I-
233;
Q-73 to C-232; Q-73 to R-231; Q-73 to F-230; Q-73 to L-229; Q-73 to T-228; Q-
73 to
V-227; Q-73 to L-226; Q-73 to S-225; Q-73 to L-224; Q-73 to E-223; Q-73 to D-
222;
Q-73 to G-221; Q-73 to F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-
73 to
K-216; Q-73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to L-
211;
Q-73 to H-210; Q-73 to G-209; Q-73 to M-208; Q-73 to A-207; Q-73 to Y-206; Q-
73 to
T-205; Q-73 to K-204; Q-73 to D-203; Q-73 to T-202; Q-73 to Y-201; Q-73 to L-
200;
Q-73 to V-199; Q-73 to Q-198; Q-73 to G-197; Q-73 to Y-196; Q-73 to I-195; Q-
73 to
F-194; Q-73 to F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-
189;
Q-73 to K-188; Q-73 to V-187; Q-73 to L-186; Q-73 to I-185; Q-73 to K-184; Q-
73 to
N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180; Q-73 to E-179; Q-73 to L-
178;
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Q-73 to A-177; Q-73 to S-176; Q-73 to G-175; Q-73 to R-174; Q-73 to K-173; Q-
73 to
F-172; Q-73 to S-171; Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-
167;
Q-73 to V-166; Q-73 to F-165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162; Q-
73 to
G-161; Q-73 to K-160; Q-73 to Q-159; Q-73 to I-158; Q-73 to T-157; Q-73 to P-
156;
Q-73 to T-155; Q-73 to E-154; Q-73 to S-153; Q-73 to D-152; Q-73 to A-151; Q-
73 to
I-150; Q-73 to L-149; Q-73 to Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-
145;
Q-73 to Q-144; Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E-140; Q-
73 to
E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-
134;
Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-
73 to
N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-
123;
Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-
73 to
P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-
112; Q-73
to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to
P-106;
Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-
73 to
A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-
73 to
G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-
73 to
A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-
73 to
E-82; Q-73 to A-81; Q-73 to R-80; and Q-73 to L-79 of SEQ ID N0:3228. The
present
invention is also directed to antibodies that bind BLyS polypeptides
comprising, or
alternatively, consisting of, a contiguous sequence of amino acid residues at
least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of
BLyS polypeptides described above.
[0127] The invention also provides antibodies that bind polypeptides having
one or
more amino acids deleted from both the amino and the carboxyl termini of the
predicted
extracellular domain of BLyS, which may be described generally as having
residues n2-m2
of SEQ ID N0:3228 where n2 and m2 are integers as defined above.
[0128] In another embodiment, antibodies of the present invention bind
polypeptides
consisting of a portion of the extracellular domain of the BLyS amino acid
sequence
encoded by the cDNA plasmid contained in the deposit having ATCC accession no.
97768, where this portion excludes from 1 to about 206 amino acids from the
amino
terminus of the extracellular domain of the amino acid sequence encoded by the
cDNA
plasmid contained in the deposit having ATCC accession no. 97768, or from 1 to
about
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206 amino acids from the carboxy terminus of the extracellular domain of the
amino acid
sequence encoded by the cDNA plasmid contained in the deposit having ATCC
accession
no. 97768, or. any combination of the above amino terminal and carboxy
terminal
deletions, of the entire extracellular domain of the amino acid sequence
encoded by the
cDNA plasmid contained in the deposit having ATCC accession no. 97768.
[0129] As mentioned above, even if deletion of one or more amino acids from
the
N-terminus of a polypeptide results in modification or loss of one or more
functional
activities (e.g., biological activity) of the polypeptide, other functions or
biological
activities rnay still be retained. Thus, the ability of a shortened BLyS
mutein to induce
and/or bind to antibodies which recognize the full-length or mature forms or
the
extracellular domain of the polypeptide generally will be retained when less
than the
majority of the residues of the full-length or mature or extracellular domain
of the
polypeptide are removed from the N-terminus. Whether a particular polypeptide
lacking
N-terminal residues of a complete polypeptide retains such immunologic
activities can
readily be determined by routine methods described herein and otherwise known
in the art.
It is not unlikely that a BLyS mutein with a large number of deleted N-
terminal amino
acid residues may retain some functional (e.g., biological or immunogenic)
activities. In
fact, peptides composed of as few as six BLyS amino acid residues may often
evoke an
immune response.
[0130] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the
predicted full-length amino acid sequence of the BLyS shown in SEQ ID N0:3228,
up to
the glycine residue at position number 280 of the sequence shown SEQ ID
NO:3228 and
polynucleotides encoding such polypeptides. In particular, the present
invention provides
antibodies that bind polypeptides comprising the amino acid sequence of
residues n3-285
of the sequence shown in SEQ ID N0:3228, where n3 is an integer in the range
of the
amino acid position of amino acid residues 1 to 280 of the amino acid sequence
in SEQ
ID N0:3228.
[0131] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the group
consisting of residues of D-2 to L-285; D-3 to L-285; S-4 to L-285; T-5 to L-
285; E-6 to
L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-11 to L-285;
L-12 to
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L-285; T-13 to L-285; S-14 to L-285; C-15 to L-285; L-16 to L-285; K-17 to L-
285; K-18
to L-285; R-19 to L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K-23 to
L-285;
L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to L-285; S-
29 to
L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285; R-33 to L-285; K-34 to L-
285; E-35
to L-285; S-36 to L-285; P-37 to L-285; S-38 to L-285; V-39 to L-285; R-40 to
L-285;
S-41 to L-285; S-42 to L-285; K-43 to L-285; D-44 to L-285; G-45 to L-285; K-
46 to
L-285; L-47 to L-285; L-48 to L-285; A-49 to L-285; A-50 to L-285; T-51 to L-
285; L-52
to L-285; L-53 to L-285; L-54 to L-285; A-55 to L-285; L-56 to L-285; L-57 to
L-285;
S-58 to L-285; C-59 to L-285; C-60 to L-285; L-61 to L-285; T-62 to L-285; V-
63 to
L-285; V-64 to L-285; S-65 to L-285; F-66 to L-285; Y-67 to L-285; Q-68 to L-
285; V-69
'to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to L-285; G-74 to
L-285;
D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-
80 to
L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 taL-
285; H-86
to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to
L-285;
P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-
97 to
L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to
L-285;
E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-
285; V-108
to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-
113 to
L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118
to L-285;
A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-
285; N-124
to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-
129 to
L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134
to
L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139
to
L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144
to
L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149
to
L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154
to L-285;
T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-
285; K-160
to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-
165 to
L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170
to
L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175
to L-285;
S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-
285; K-181 w
to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-
186 to
CA 02467521 2004-05-14
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L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191
to
L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196
to L-285;
G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-
285; T-202
to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-
207 to
L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212
to
L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217
to
L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222
to
L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227
to L-285;
T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-
285; I-233
to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-
238 to
L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243
to L-285;
S-244 to L=285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-
285; G-249
to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-
254 to
L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259
to
L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264
to L-285;
R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-
285; I-270
to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-
275 to
L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-
280 to
L-285 of SEQ ID N0:3228. The present invention is also directed to antibodies
that bind
BLyS polypeptides comprising, or alternatively, consisting of, a contiguous
sequence of
amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of BLyS polypeptides described above.
[0132] Also as mentioned above, even if deletion of one or more amino acids
from the
C-terminus of a protein results in modification or loss of one or more
functional activities
(e.g., biological activity) of the protein, other functional activities may
still be retained.
Thus, the ability of a shortened BLyS mutein to induce and/or bind to
antibodies which
recognize the complete or mature form or the extracellular domain of the
polypeptide
generally will be retained when less than the majority of the residues of the
complete or
mature form or the extracellular domain of the polypeptide are removed from
the
C-terminus. Whether a particular polypeptide lacking C-terminal residues of a
complete
polypeptide retains such immunologic activities can readily be determined by
routine
methods described herein and otherwise known in the art. It is not unlikely
that a BLyS
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mutein with a large number of deleted C-terminal amino acid residues may
retain some
functional (e.g., biological or immunogenic) activities. In fact, peptides
composed of as
few as six BLyS amino acid residues may often evoke an immune response.
[0133] Accordingly, the present invention further provides in another
embodiment,
antibodies that bind polypeptides having one or more residues deleted from the
carboxy
terminus of the amino acid sequence of the BLyS shown in SEQ ID N0:3228, up to
the
glutamic acid residue at position number 6, and polynucleotides encoding such
polypeptides. In particular, the present invention provides antibodies that
bind
polypeptides comprising the amino acid sequence of residues 1-m3 of SEQ ID
N0:3228,
where m3 is an integer in the range of the amino acid position of amino acid
residues
6-284 of the amino acid sequence in SEQ ID N0:3228.
[0134] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the group
consisting of residues M-1 to L-284; M-1 to K-283; M-1 to L-282; M-1 to A-281;
M-1 to
G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; M-1 to V-276; M-1 to D-275; M-
1 to
G-274; M-1 to D-273; M-1 to L-272; M-1 to S-271; M-1 to I-270; M-1 to Q-269; M-
1 to
A-268; M-1 to N-267; M-1 to E-266; M-1 to R-265; M-1 to P-264; M-1 to I-263; M-
1 to
A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258; M-1 to D-257; M-
1 to
G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253; M-1 to K-252; M-1 to A-251; M-
1 to
I-250; M-1 to G-249; M-1 to A-248; M-1 to S-247; M-1 to Y-246; M-1 to C-245; M-
1 to
S-244; M-1 to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T-239; M-
1 to
E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to Q-234; M-1 to I-233; M-
1 to
C-232; M-1 to R-231; M-1 to F-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-
1 to
L-226; M-1 to S-225; M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-
1 to
F-220; M-1 to V-219; M-1 to H-218; M-1 to V-217; M-1 to K-216; M-1 to K-215; M-
1 to
R-214; M-1 to Q-213; M-1 to I-212; M-1 to L-211; M-1 to H-210; M-1 to G-209; M-
1 to
M-208; M-1 to A-207; M-1 to Y-206; M-1 to T-205; M-1 to K-204; M-1 to D-203; M-
1 to
T-202; M-1 to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197; M-
1 to
Y-196; M-1 to I-195; M-1 to F-194; M-1 to F-193; M-1 to Y-192; M-1 to G-191; M-
1 to
T-190; M-1 to E-189; M-1 to K-188; M-1 to V-187; M-1 to L-186; M-1 to I-185; M-
1 to
K-184; M-1 to N-183; M-1 to E-182; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-
1 to
L-178; M-1 to A-177; M-1 to S-176; M-1 to G-175; M-1 to R-174; M-1 to K-173; M-
1 to
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F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169; M-1 to W-168; M-1 to P-167; M-
1 to
V-166; M-1 to F-165; M-1 to T-164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-
1 to
K-160; M-1 to Q-159; M-1 to I-158; M-1 to T-157; M-1 to P-156; M-1 to T-155; M-
1 to
E-154; M-1 to S-153; M-1 to D-152; M-1 to A-151; M-1 to I-150; M-1 to L-149; M-
1 to
Q-148; M-1 to L-147; M-1 to C-146; M-1 to D-145; M-1 to Q-144; M-1 to T-143; M-
1 to
V-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-
1 to
Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-
1 to
R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-
1 to
N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-
1 to
P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-
1 to
L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-
1 to
P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-
1 to
A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to
G-94;
M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88;
M-1 to
H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to
A-81; ,
M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75;
M-1 to
G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to
Q-68;
M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62;
M-1 to
L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to
A-55;
M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49;
M-1 to
L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to
S-42;
M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36;
M-1 to
E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to
S-29;
M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23;
M-1 to
M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to
L-16;
M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10;
M-1 to
Q-9; M-1 to E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID N0:3228. The present
invention
is also directed to antibodies that bind BLyS polypeptides comprising, or
alternatively,
consisting of, a contiguous sequence of amino acid residues at least 80%, 85%,
90%, 92%,
95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS
polypeptides
described above.
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[0135] The invention also provides antibodies that bind polypeptides having
one or
more amino acids deleted from both the amino and the carboxyl termini of a
BLyS
polypeptide, which may be described generally as having residues n3-m3 of SEQ
ll~
N0:3228, where n3 and m3 are integers as defined above.
[0136] Furthermore, since the predicted extracellular domain of the BLyS
polypeptide
of SEQ ID N0:3229 may itself elicit functional activity (e.g., biological
activity),
deletions of N- and C-terminal amino acid residues from the predicted
extracellular region
of the polypeptide at positions Gln-73 to Leu-266 of SEQ ID N0:3229 may retain
some
functional activity, such as, for example, ligand binding, to stimulation of
lymphocyte
(e.g., B cell) proliferation, differentiation, and/or activation, modulation
of cell
replication, modulation of target cell activities and/or immunogenicity.
However, even if
deletion of one or more amino acids from the N-terminus of the predicted
extracellular
domain of a BLyS polypeptide results in modification or loss of one or more
functional
activities of the polypeptide, other functional activities rnay still be
retained. Thus, the
ability of the shortened polypeptides to induce and/or bind to antibodies
which recognize
the complete or mature or extracellular domains of the polypeptides generally
will be
retained when less than the majority of the residues of the complete or mature
or
extracellular domains of the polypeptides are removed from the N-terminus.
Whether a
particular polypeptide lacking N-terminal residues of a complete polypeptide
retains such
immunologic activities can readily be determined by routine methods described
herein and
otherwise known in the art.
[0137] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the amino
acid sequence of BLyS shown in SEQ ID N0:3229, up to the glycine residue at
position
number 261. In particular, the present invention provides antibodies that bind
polypeptides comprising the amino acid sequence of residues n4-266 of SEQ ll~
N0:3229,
where n4 is an integer in the range of the amino acid position of amino acid
residues
73-261 of the amino acid sequence in SEQ ID N0:3229, and 261 is the position
of the first
residue from the N-terminus of the predicted extracellular domain BLyS
polypeptide
(shown in SEQ ID N0:3229).
[0138] More in particular, in certain embodiments, the invention provides
antibodies
that bind polypeptides comprising, or alternatively consisting of, an amino
acid sequence
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WO 03/055979 PCT/US02/36496
selected from the group consisting of residues of Q-73 to L-266; G-74 to L-
266; D-75 to
L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-
266; A-81
to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to
L-266;
H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-
92 to
L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-
266; P-98
to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103
to
L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108
to
L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113
to
L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118
to L-266;
A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to Ir266; G-123 to L-
266; N-124
to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-
129 to
L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134
to
L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139
to
L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144
to L-266;
T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-
266; L-150
to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-
155 to
L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160
to L-266;
E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-
266; I-166
to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-
171 to
L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176
to L-266;
Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-
266;
Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-
266; Y-187
to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-
192 to
L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197
to L-266;
V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-
266;
D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-
266; V-208
to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-
213 to
L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218
to L-266;
E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-
266; N-224
to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-
229 to
L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234
to L-266;
E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-
266; L-240
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-
245 to
L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250
to
L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255
to L-266;
D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-
266; and
G-261 to L-266 of SEQ ID N0:3229. The present invention is also directed to
antibodies
that bind BLyS polypeptides comprising, or alternatively, consisting of, a
contiguous
sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or
99% identical to the amino acid sequence of BLyS polypeptides described above.
[0139] Similarly, deletions of C-terminal amino acid residues of the predicted
extracellular domain of BLyS up to the leucine residue at position 79 of SEQ
ID N0:3229
may retain some functional activity, such as, for example, ligand binding, the
ability to
stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or
activation,
modulation of cell replication, modulation of target cell activities andlor
immunogenicity.
Polypeptides having further C-terminal deletions including Leu-79 of SEQ ID
N0:3229
would not be expected to retain biological activities.
[0140] However, even if deletion of one or more amino acids from the C-
terminus of a
polypeptide results in modification or loss of one or more functional
activities (e.g.,
biological activity) of the polypeptide, other functional activities may still
be retained.
Thus, the ability of the shortened polypeptide to induce and/or bind to
antibodies which
recognize the complete, mature or extracellular forms of the polypeptide
generally will be
retained when less than the majority of the residues of the complete, mature
or
extracellular forms of the polypeptide are removed from the C-terminus.
Whether a
particular polypeptide lacking C-terminal residues of the predicted
extracellular domain
retains such immunologic activities can readily be determined by routine
methods
described herein and otherwise known in the art.
[0141] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues from the carboxy terminus of the
amino acid
sequence of the predicted extracellular domain of BLyS shown in SEQ ID
N0:3229, up to
the leucine residue at position 79 of SEQ ID N0:3229. In particular, the
present invention
provides antibodies that bind polypeptides having the amino acid sequence of
residues
73-m4 of the amino acid sequence in SEQ ID NO:3229, where m4 is any integer in
the
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range of the amino acid position of amino acid residues 79-265 of the amino
acid
sequence in SEQ ID N0:3229.
[0142] More in particular, in certain embodiments, the invention provides
antibodies
that bind polypeptides comprising, or alternatively consisting of, an amino
acid sequence
selected from the group consisting of residues Q-73 to L-265; Q-73 to K-264; Q-
73 to
L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-
258;
Q-73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-
73 to
S-252; Q-73 to I-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-
247;
Q-73 to R-246; Q-73 to P-245; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-
73 to
Q-241; Q-73 to L-240; Q-73 to E-239; Q-73 to D-238; Q-73 to G-237; Q-73 to E-
236;
Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to A-232; Q-73 to I-231; Q-
73 to
G-230; Q-73 to A-229; Q-73 to S-228; Q-73 to Y-227; Q-73 to C-226; Q-73 to S-
225;
Q-73 to N-224; Q-73 to N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-
73 to
E-219; Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; Q-73 to I-
214;
Q-73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210; Q-73 to T-209; Q-
73 to
V-208; Q-73 to L-207; Q-73 to S-206; Q-73 to L-205; Q-73 to E-204; Q-73 to D-
203;
Q-73 to G-202; Q-73 to F-201; Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-
73 to
K-197; Q-73 to K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-
192;
Q-73 to H-191; Q-73 to G-190; Q-73 to Q-7389; Q-73 to A-188; Q-73 to Y-187; Q-
73 to
T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to T-183; Q-73 to Y-182; Q-73 to L-
181;
Q-73 to V-180; Q-73 to Q-179; Q-73 to G-178; Q-73 to Y-177; Q-73 to I-176; Q-
73 to
F-175; Q-73 to F-174; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-
170;
Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to K-165; Q-
73 to
N-164; Q-73 to E-163; Q-73 to K-162; Q-73 to E-161; Q-73 to E-160; Q-73 to L-
159;
Q-73 to A-158; Q-73 to S-157; Q-73 to G-156; Q-73 to R-155; Q-73 to K-154; Q-
73 to
F-153; Q-73 to S-152; Q-73 to L-151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-
148;
Q-73 to V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143; Q-
73 to
G-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-
137;
Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-
73 to
N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-
126;
Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-
73 to
P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-
115;
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Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-
73 to
T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-
104;
Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to I~-99; Q-
73 to
P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-
73 to
P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-
73 to
H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-
73 to
R-80; Q-73 to L-79; and Q-73 to S-78 of SEQ ID NO:3229. The present invention
is also
directed to antibodies that bind BLyS polypeptides comprising, or
alternatively, consisting
of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%,
95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides
described above.
[0143] The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini of the predicted
extracellular
domain of BLyS, which may be described generally as having residues n4-m4 of
SEQ ID
N0:3229 where n4 and mø are integers as defined above.
[0144] In another embodiment, antibodies of the present invention bind
polypeptides
consisting of a portion of the extracellular domain of the BLyS amino acid
sequence
encoded by the cDNA clone contained in the deposit having ATCC Accession No.
203518, where this portion excludes from 1 to about 260 amino acids from the
amino
terminus of the extracellular domain of the amino acid sequence encoded by
cDNA clone
contained in the deposit having ATCC Accession No. 203518, or from 1 to about
187
amino acids from the carboxy terminus of the extracellular domain of the amino
acid
sequence encoded by cDNA clone contained in the deposit having ATCC Accession
No.
203518, or any combination of the above amino terminal and carboxy terminal
deletions,
of the entire extracellular domain of the amino acid sequence encoded by the
cDNA clone
contained in the deposit having ATCC Accession No. 203518.
[0145] As mentioned above, even if deletion of one or more amino acids from
the
N-terminus of a polypeptide results in modification or loss of one or more
functional
activities (e.g., biological activity) of the polypeptide, other functional
activities may still
be retained. Thus, the ability of a shortened BLyS polypeptide to induce
and/or bind to
antibodies which recognize the full-length or mature forms or the
extracellular domain of
the polypeptide generally will be retained when less than the majority of the
residues of
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the full-length or mature or extracellular domain of the polypeptide are
removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal residues of a
complete
polypeptide retains such immunologic activities can readily be determined by
routine
methods described herein and otherwise known in the art. It is not unlikely
that a BLyS
mutein with a large number of deleted N-terminal amino acid residues may
retain
functional (e.g., immunogenic) activities. In fact, peptides composed of as
few as six
BLyS amino acid residues may often evoke an immune response.
[0146] Accordingly, the present invention further provides antibodies that
bind
polypeptides having one or more residues deleted from the amino terminus of
the
predicted full-length amino acid sequence of the BLyS polypeptide shown in SEQ
ID
N0:3229, up to the glycine residue at position number 261 of the sequence
shown SEQ ID
N0:3229 and polynucleotides encoding such polypeptides. In particular, the
present
invention provides antibodies that bind polypeptides comprising the amino acid
sequence
of residues ns-266 of the sequence shown in SEQ ID N0:3229, where ns is an
integer in
the range of the amino acid position of amino acid residues 1 to 261 of the
amino acid
sequence in SEQ ID NO:3229.
[0147] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the group
consisting of residues of D-2 to L-266; D-3 to L-266; S-4 to L-266; T-5 to L-
266; E-6 to
L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-11 to L-266;
L-12 to
L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L-266; K-l7 to L-
266; K-18
to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to
L-266;
L-24 to L-266; K-25 to L-266; E-26 to L-266; C-27 to L-266; V-28 to L-266; S-
29 to
L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266; R-33 to L-266; K-34 to L-
266; E-35
to L-266; S-36 to L-266; P-37 to L-266; S-38 to L-266; V-39 to L-266; R-40 to
L-266;
S-41 to L-266; S-42 to L-266; K-43 to L-266; D-44 to L-266; G-45 to L-266; K-
46 to
L-266; L-47 to L-266; L-48 to L-266; A-49 to L-266; A-50 to L-266; T-51 to L-
266; L-52
to L-266; L-53 to L-266; L-54 to L-266; A-55 to L-266; L-56 to L-266; L-57 to
L-266;
S-58 to L-266; C-59 to L-266; C-60 to L-266; L-61 to L-266; T-62 to L-266; V-
63 to
L-266; V-64 to L-266; S-65 to L-266; F-66 to L-266; Y-67 to L-266; Q-68 to L-
266; V-69
to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to L-266; G-74 to
L-266;
D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-
80 to
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L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-
266; H-86
to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to
L-266;
P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-
97 to
L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to
L-266;
E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-
266; V-108
to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-
113 to
L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118
to L-266;
A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-
266; N-124
to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-
129 to
L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134
to
L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139
to
L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144
to L-266;
T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-
266; L-150
to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-
155 to
L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160
to L-266;
E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-
266; I-166
to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-
171 to
L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176
to L-266;
Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-
266;
Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-
266; Y-187
to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-
192 to
L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197
to L-266;
V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-
266;
D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-
266; V-208
to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-
213 to
L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218
to L-266;
E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-
266; N-224
to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-
229 to
L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234
to L-266;
E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-
266; L-240
to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-
245 to
L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250
to
CA 02467521 2004-05-14
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L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255
to L-266;
D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259,to L-266; F-260 to L-
266; and
G-261 to L-266 of SEQ ID N0:3229. The present invention is also directed to
antibodies
that bind BLyS polypeptides comprising, or alternatively, consisting of, a
contiguous
sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or
99% identical to the amino acid sequence of BLyS polypeptides described above.
[0148] Also as mentioned above, even if deletion of one or more amino acids
from the
C-terminus of a protein results in modification or loss of one or more
functional activities
(e.g., biological activities) of the protein, other functional activities may
still be retained.
Thus, the ability of a shortened BLyS mutein to induce andlor bind to
antibodies which
recognize the complete or mature form or the extracellular domain of the
polypeptide
generally will be retained when less than the majority of the residues of the
complete or
mature form or the extracellular domain of the polypeptide are removed from
the
C-terminus. Whether a particular polypeptide lacking C-terminal residues of a
complete
polypeptide retains such immunologic activities can readily be determined by
routine
methods described herein and otherwise known in the art. It is not unlikely
that a BLyS
mutein with a large number of deleted C-terminal amino acid residues may
retain some
functional (e.g., immunogenic) activities. In fact, peptides composed of as
few as six
BLyS amino acid residues may often evoke an immune response.
[0149] Accordingly, the present invention further provides in another
embodiment,
antibodies that bind polypeptides having one or more residues deleted from the
carboxy
terminus of the amino acid sequence of the BLyS shown in SEQ ID N0:3229, up to
the
glutamic acid residue at position number 6, and polynucleotides encoding such
polypeptides. In particular, the present invention provides antibodies that
bind
polypeptides comprising the amino acid sequence of residues 1-ms of SEQ ID
N0:3229,
where ms is an integer in the range of the amino acid position of amino acid
residues 6 to
265 in the amino acid sequence of SEQ ID N0:3229.
[0150] More in particular, the invention provides antibodies that bind
polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the group
consisting of residues M-1 to L-265; M-1 to I~-264; M-1 to L-263; M-1 to A-
262; M-1 to
G-261; M-1 to F-260; M-1 to F-259; M-1 to T-258; M-1 to V-257; M-1 to D-256; M-
1 to
G-255; M-1 to D-254; M-1 to L-253; M-1 to S-252; M-1 to I-251; M-1 to Q-250; M-
1 to
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A-249; M-1 to N-248; M-1 to E-247; M-1 to R-246; M-1 to P-245; M-1 to I-244; M-
1 to
A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1 to D-238; M-
1 to
G-237; M-1 to E-236; M-1 to E-235; M-1 to L-234; M-1 to K-233; M-1 to A-232; M-
1 to
I-231; M-1 to G-230; M-1 to A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226; M-
1 to
S-225; M-1 to N-224; M-1 to N-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-
1 to
E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to Q-215; M-1 to I-214; M-
1 to
C-213; M-1 to R-212; M-1 to F-211; M-1 to L-210; M-1 to T-209; M-1 to V-208; M-
1 to
L-207; M-1 to S-206; M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-
1 to
F-201; M-1 to V-200; M-1 to H-199; M-1 to V-198; M-1 to K-197; M-1 to K-196; M-
1 to
R-195; M-1 to Q-194; M-1 to I-193; M-1 to L-192; M-1 to H-191; M-1 to G-190; M-
1 to
M-189; M-1 to A-188; M-1 to Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-
1 to
T-183; M-1 to Y-182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178; M-
1 to
Y-177; M-1 to I-176; M-1 to F-175; M-1 to F-174; M-1 to Y-173; M-1 to G-172; M-
1 to
T-171; M-1 to E-170; M-1 to K-169; M-1 to V-168; M-1 to L-167; M-1 to I-166; M-
1 to
K-165; M-1 to N-164; M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-
1 to
L-159; M-1 to A-158; M-1 to S-157; M-1 to G-156; M-1 to R-155; M-1 to K-154; M-
1 to
F-153; M-1 to S-152; M-1 to L-151; M-1 to L-150; M-1 to W-149; M-1 to P-148; M-
1 to
V-147; M-1 to F-146; M-1 to T-145; M-1 to Y-144; M-1 to S-143; M-1 to G-142; M-
1 to
T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-
1 to
V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-
1 to
5-129; M-1 to N-128; M-l to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-
1 to
G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-
1 to
P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-
1 to
G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-
1 to
A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-
1 to
K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to
A-93;
M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87;
M-1 to
H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to
R-80;
M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74;
M-1 to
Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to
Y-67;
M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61;
M-1 to
C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to
L-54;
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M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48;
M-1 to
L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to
S-41;
M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35;
M-1 to
K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to
V-28;
M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22;
M-1 to
E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to
C-15;
M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-
1 to
E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID N0:3229. The present invention is
also
directed to antibodies that bind BLyS polypeptides comprising, or
alternatively, consisting
of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%,
95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides
described above.
[0151] The invention also provides antibodies that bind polypeptides having
one or
more amino acids deleted from both the amino and the carboxyl termini of a
BLyS
polypeptide, which may be described generally as having residues n5-ms of SEQ
ID
N0:3229, where ns and ms are integers as defined above.
[0152] In additional embodiments, the present invention provides antibodies
that bind
polypeptides comprising the amino acid sequence of residues 134-m6 of SEQ ID
N0:3228, where m6 is an integer from 140 to 285, corresponding to the position
of the
amino acid residue in SEQ ID N0:3228. For example, the invention provides
antibodies
that bind polypeptides comprising, or alternatively consisting of, an amino
acid sequence
selected from the group consisting of residues A-134 to Leu-285; A-134 to L-
284; A-134
to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-
134 to
F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134
to
D-273; A-134 to L-272; A-134 to S-271; A-134 to I-270; A-134 to Q-269; A-134
to
A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134
to
I-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-259; A-134
to
E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to E-254; A-134
to
L-253; A-134 to K-252; A-134 to A-251; A-134 to I-250; A-134 to G-249; A-134
to
A-248; A-134 to S-247; A-134 to Y-246; A-134 to C-245; A-134 to S-244; A-134
to
N-243; A-134 to N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134
to
E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; A-134 to Q-234; A-134
to
7s
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I-233; A-134 to C-232; A-134 to R-231; A-134 to F-230; A-134 to L-229; A-134
to
T-228; A-134 to V-227; A-134 to L-226; A-134 to S-225; A-134 to L-224; A-134
to
E-223; A-134 to D-222; A-134 to G-221; A-134 to F-220; A-134 to V-219; A-134
to
H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to R-214; A-134
to
Q-213; A-134 to I-212; A-134 to L-211; A-134 to H-210; A-134 to G-209; A-134
to
M-208; A-134 to A-207; A-134 to Y-206; A-134 to T-205; A-134 to K-204; A-134
to
D-203; A-134 to T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134
to
Q-198; A-134 to G-197; A-134 to Y-196; A-134 to I-195; A-134 to F-194; A-134
to
F-193; A-134 to Y-192; A-134 to G-191; A-134 to T-190; A-134 to E-189; A-134
to
K-188; A-134 to V-187; A-134 to L-186; A-134 to I-185; A-134 to K-184; A-134
to
N-183; A-134 to E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134
to
L-178; A-134 to A-177; A-134 to S-176; A-134 to G-175; A-134 to R-174; A-134
to
K-173; A-134 to F-172; A-134 to S-171; A-134 to L-170; A-134 to L-169; A-134
to
W-168; A-134 to P-167; A-134 to V-166; A-134 to F-165; A-134 to T-164; A-134
to
Y-163; A-134 to S-162; A-134 to G-161; A-134 to K-160; A-134 to Q-159; A-134
to
I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to E-154; A-134
to S-153;
A-134 to D-152; A-134 to A-151; A-134 to I-150; A-134 to L-149; A-134 to Q-
148; A-
134 to L-147; A-134 to C-146; A-134 to D-145; A-134 to Q-144; A-134 to T-143;
A-134
to V-142; A-134 to T-141; and A-134 to E-140 of SEQ ID NQ:3228. The present
invention is also directed to antibodies that bind BLyS polypeptides
comprising, or
alternatively, consisting of, a contiguous sequence of amino acid residues at
least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of
BLyS polypeptides described above.
[0153] In additional embodiments, antibodies of the present invention may bind
polypeptide fragments comprising, or alternatively consisting of, an amino
acid sequence
selected from the group consisting of residues: M-1 to C-15; D-2 to L-16; D-3
to K-17; S-
4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-
10 to L-24;
R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to
I-30; K-17
to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36;
K-23 to
P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-
29 to K-
43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35
to A-49;
S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to
A-55; S-42
79
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to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61;
L-48 to
T-62; A-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-
54 to Q-
68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60
to G-74;
L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to
R-80; Y-
67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-
86; Q-73
to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92;
L-79 to
A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-
85 to K-
99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104;
L-91 to
A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-
110; A-
97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-
102 to E-
116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to
G-121;
V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-
126; K-
113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131;
P-118 to
K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123
to 6-
137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to
V-142;
S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to C-146; R-133 to L-
147; A-
134 to Q-148; V-135 to L-149; Q-136 to I-150; G-137 to A-151; P-138 to D-152;
E-139 to
S-153; E-140 to E-154; T-141 to T-155; V-142 to P-156; T-143 to T-157; Q-144
to I-158;
D-145 to Q-159; C-146 to K-160; L-147 to G-161; Q-148 to S-162; L-149 to Y-
163; I-150
to T-164; A-151 to F-165; D-152 to V-166; S-153 to P-167; E-154 to W-168; T-
155 to L-
169; P-156 to L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to
R-174;
G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to L-178; F-165 to E-
179; V-166
to E-180; P-167 to K-181; W-168 to E-182; L-169 to N-183; L-170 to K-184; S-
171 to I-
185; F-172 to L-186; K-173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to
T-190;
A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to F-194; K-181 to I-
195; E-182
to Y-196; N-183 to G-197; K-184 to Q-198; I-185 to V-199; L-186 to L-200; V-
187 to Y-
201; K-188 to T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205; Y-192 to
Y-206;
F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to H-210; G-197 to L-
211; Q-
198 to I-212; V-199 to Q-213; L-200 to R-214; Y-201 to K-215; T-202 to K-216;
D-203
to V-217; K-204 to H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-
208 to
D-222; G-209 to E-223; H-210 to L-224; L-211 to S-225; I-212 to L-226; Q-213
to V-
227; R-214 to T-228; K-215 to L-229; K-216 to F-230; V-217 to R-231; H-218 to
C-232;
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V-219 to I-233; F-220 to Q-234; G-221 to N-235; D-222 to M-236; E-223 to P-
237; L-224
to E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to N-242; L-
229 to N-
243; F-230 to S-244; R-231 to C-245; C-232 to Y-246; I-233 to S-247; Q-234 to
A-248;
N-235 to G-249; M-236 to I-250; P-237 to A-251; E-238 to K-252; T-239 to L-
253; L-240
to E-254; P-241 to E-255; N-242 to G-256; N-243 to D-257; S-244 to E-258; C-
245 to L-
259; Y-246 to Q-260; S-247 to L-261; A-248 to A-262; G-249 to I-263; I-250 to
P-264;
A-251 to R-265; K-252 to E-266; L-253 to N-267; E-254 to A-268; E-255 to Q-
269; 6-
256 to I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to G-274;
L-261 to
D-275; A-262 to V-276; I-263 to T-277; P-264 to F-278; R-265 to F-279; E-266
to G-280;
N-267 to A-281; A-268 to L-282; Q-269 to K-283; I-270 to L-284; and S-271 to L-
285 of
SEQ ID N0:3228. The present invention is also directed to antibodies that bind
BLyS
polypeptides comprising, or alternatively, consisting of, a contiguous
sequence of amino
acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical
to the
amino acid sequence of BLyS polypeptides described above.
[0154] In additional embodiments, antibodies of the present invention may bind
polypeptide fragments comprising, or alternatively consisting of, an amino
acid sequence
selected from the group consisting of residues: M-1 to C-15; D-2 to L-16; D-3
to K-17; S-
4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-
10 to L-24;
R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to
I-30; K-17
to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36;
K-23 to
P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-
29 to K-
43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35
to A-49;
S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to
A-55; S-42
to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61;
L-48 to
T-62; A-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-
54 to Q-
68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60
to G-74;
L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to
R-80; Y-
67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-
86; Q-73
to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92;
L-79 to
A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-
85 to K-
99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104;
L-91 to
A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-
110; A-
81
CA 02467521 2004-05-14
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97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-
102 to E-
116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to
G-121;
V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-
126; K-
113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131;
P-118 to
K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123
to 6-
137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to
G-142;
S-129 to S-143; R-130 to Y-144; N-131 to T-145; K-132 to F-146; R-133 to V-
147; A-
134 to P-148; V-135 to W-149; Q-136 to L-150; G-137 to L-151; P-138 to S-152;
E-139
to F-153; E-140 to K-154; T-141 to R-155; G-142 to G-156; S-143 to S-157; Y-
144 to A-
158; T-145 to L-159; F-146 to E-160; V-147 to E-161; P-148 to K-162; W-149 to
E-163;
L-150 to N-164; L-151 to K-165; S-152 to I-166; F-153 to L-167; K-154 to V-
168; R-155
to K-169; G-156 to E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-
160 to F-
174; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to G-178; K-165 to
Q-179;
I-166 to V-180; L-167 to L-181; V-168 to Y-182; K-169 to T-183; E-170 to D-
184; T-171
to K-185; G-172 to T-186; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-
176 to 6-
190; Y-177 to H-191; G-178 to L-192; Q-179 to I-193; V-180 to Q-194; L-181 to
R-195;
Y-182 to K-196; T-183 to K-197; D-184 to V-198; K-185 to H-199; T-186 to V-
200; Y-
187 to F-201; A-188 to G-202; M-189 to D-203; G-190 to E-204; H-191 to L-205;
L-192
to S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to L-210; K-
197 to F-
211; V-198 to R-212; H-199 to C-213; V-200 to I-214; F-201 to Q-215; G-202 to
N-216;
D-203 to M-217; E-204 to P-218; L-205 to E-219; S-206 to T-220; L-207 to L-
221; V-208
to P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to C-226; C-
213 to Y-
227; I-214 to S-228; Q-215 to A-229; N-216 to G-230; M-217 to I-231; P-218 to
A-232;
E-219 to K-233; T-220 to L-234; L-221 to E-235; P-222 to E-236; N-223 to G-
237; N-224
to D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; S-228 to L-242; A-
229 to A-
243; G-230 to I-244; I-231 to P-245; A-232 to R-246; K-233 to E-247; L-234 to
N-248; E-
235 to A-249; E-236 to Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253;
L-240 to
D-254; Q-241 to G-255; L-242 to D-256; A-243 to V-257; I-244 to T-258; P-245
to F-
259; R-246 to F-260; E-247 to G-261; N-248 to A-262; A-249 to L-263; Q-250 to
K-264;
I-251 to L-265; and S-252 to L-266 of SEQ ID N0:3229. The present invention is
also
directed to antibodies that bind BLyS polypeptides comprising, or
alternatively, consisting
of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%,
95%,
82
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96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides
described above.
[0155] In additional embodiments, antibodies of the present invention may bind
polypeptide fragments comprising, or alternatively consisting of, an amino
acid sequence
selected from the group consisting of residues: M-1 to F-15; D-2 to C-16; E-3
to S-17; S-4
to E-18; A-5 to K-19; K-6 to G-20; T-7 to E-21; L-8 to D-22; P-9 to M-23; P-10
to K-24;
P-11 to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to
I-30; S-17
to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35; D-22 to E-36;
M-23 to
G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to I-42; P-
29 to C-
43; I-30 to R-44; T-31 to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35
to L-49;
E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F-40 to L-54; G-41 to
L-55; I-
42 to A-56; C-43 to L-57; R-44 to L-58; D-45 to S-59; G-46 to S-60; R-47 to S-
61; L-48
to F-62; L-49 to T-63; A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67;
L-54 to
Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59 to L-73; S-
60 to Q-
74; S-61 to A-75; F-62 to D-76; T-63 to L-77; A-64 to M-78; M-65 to N-79; S-66
to L-80;
L-67 to R-81; Y-68 to M-82; Q-69 to E-83; L-70 to L-84; A-71 to Q-85; A-72 to
S-86; L-
73 to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91; M-78 to T-
92; N-79
to P-93; L-80 to A-94; R-81 to A-95; M-82 to A-96; E-83 to G-97; L-84 to A-98;
Q-85 to
P-99; S-86 to E-100; Y-87 to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-
104; A-91
to V-105; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-95 to T-109; A-96 to
P-110;
G-97 to A-111; A-98 to A-112; P-99 to P-113; E-100 to R-114; L-101 to P-115; T-
102 to
H-116; A-103 to N-117; G-104 to S-118; V-105 to S-119; K-106 to R-120; L-107
to 6-
121; L-108 to H-122; T-109 to R-123; P-110 to N-124; A-111 to R-125; A-112 to
R-126;
P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to G-130; N-117 to P-
131; S-118
to E-132; S-119 to E-133; R-120 to T-134; G-121 to E-135; H-122 to Q-136; R-
123 to D-
137; N-124 to V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to
A-142;
Q-129 to P-143; G-130 to P-144; P-131 to A-145; E-132 to P-146; E-133 to C-
147; T-134
to L-148; E-135 to P-149; Q-136 to G-150; D-137 to C-151; V-138 to R-152; D-
139 to H-
153; L-140 to S-154; S-141 to Q-155; A-142 to H-156; P-143 to D-157; P-144 to
D-158;
A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to N-162; P-149 to L-
163; 6-
150 to R-164; C-151 to N-165; R-152 to I-166; H-153 to I-167; S-154 to Q-168;
Q-155 to
D-169; H-156 to C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160
to I-
83
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WO 03/055979 PCT/US02/36496
174; M-161 to A-175; N-162 to D-176; L-163 to S-177; R-164 to D-178; N-165 to
T-179;
I-166 to P-180; I-167 to A-181; Q-168 to L-182; D-169 to E-183; C-170 to E-
184; L-171
to K-185; Q-172 to E-186; L-173 to N-187; I-174 to K-188; A-175 to I-189; D-
176 to V-
190; S-177 to V-191; D-178 to R-192; T-179 to Q-193; P-180 to T-194; A-181 to
G-195;
L-182 to Y-196; E-183 to F-197; E-184 to F-198; K-185 to I-199; E-186 to Y-
200; N-187
to S-201; K-188 to Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-
192 to T-
206; Q-193 to D-207; T-194 to P-208; G-195 to I-209; Y-196 to F-210; F-197 to
A-211;
F-198 to M-212; I-199 to G-213; Y-200 to H-214; S-201 to V-215; Q-202 to I-
216; V-203
to Q-217; L-204 to R-218; Y-205 to K-219; T-206 to K-220; D-207 to V-221; P-
208 to H-
222; I-209 to V-223; F-210 to F-224; A-211 to G-225; M-212 to D-226; G-213 to
E-227;
H-214 to L-228; V-215 to S-229; I-216 to L-230; Q-217 to V-231; R-218 to T-
232; K-219
to L-233; K-220 to F-234; V-221 to R-235; H-222 to C-236; V-223 to I-237; F-
224 to Q-
238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to K-242; S-229 to
T-243; .
L-230 to L-244; V-231 to P-245; T-232 to N-246; L-233 to N-247; F-234 to S-
248; R-235
to C-249; C-236 to Y-250; I-237 to S-251; Q-238 to A-252; N-239 to G-253; M-
240 to I-
254; P-241 to A-255; K-242 to R-256; T-243 to L-257; L-244 to E-258; P-245 to
E-259;
N-246 to G-260; N-247 to D-261; S-248 to E-262; C-249 to I-263; Y-250 to Q-
264; S-251
to L-265; A-252 to A-266; G-253 to I-267; I-254 to P-268; A-255 to R-269; R-
256 to E-
270; L-257 to N-271; E-258 to A-272; E-259 to Q-273; G-260 to I-274; D-261 to
S-275;
E-262 to R-276; I-263 to N-277; Q-264 to G-278; L-265 to D-279; A-266 to D-
280; I-267
to T-281; P-268 to F-282; R-269 to F-283; E-270 to G-284; N-271 to A-285; A-
272 to L-
286; Q-273 to K-287; I-274 to L-288; and S-275 to L-289 of SEQ ID N0:38. The
present
invention is also directed to antibodies that bind BLyS polypeptides
comprising, or
alternatively, consisting of, a contiguous sequence of amino acid residues at
least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of
BLyS polypeptides described above.
[0156] It will be recognized by one of ordinary skill in the art that some
amino acid
sequences of the BLyS polypeptides can be varied without significant effect of
the
structure or function of the polypeptide. If such differences in sequence are
contemplated,
it should be remembered that there will be critical areas on the polypeptide
which
determine activity.
84
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WO 03/055979 PCT/US02/36496
[0157] Thus, the invention further includes antibodies that bind variations of
BLyS
polypeptides which show BLyS polypeptide functional activity (e.g., biological
activity)
or which include regions of BLyS polypeptide such as the polypeptide fragments
described herein. Such mutants include deletions, insertions, inversions,
repeats, and type
substitutions selected according to general rules known in the art so as have
little effect on
activity. For example, guidance concerning how to make phenotypically silent
amino acid
substitutions is provided in Bowie, J. U. et al., "Deciphering the Message in
Protein
Sequences: Tolerance to Amino Acid Substitutions," Scie~ece 247:1306-1310
(1990),
wherein the authors indicate that there are two main approaches for studying
the tolerance
of an amino acid sequence to change. The first method relies on the process of
evolution,
in which mutations are either accepted or rejected by natural selection. The
second
approach uses genetic engineering to introduce amino acid changes at specific
positions of
a cloned gene and selections or screens to identify sequences that maintain
functionality.
[0158] As the authors state, these studies have revealed that proteins are
surprisingly
tolerant of amino acid substitutions. The authors further indicate which amino
acid
changes are likely to be permissive at a certain position of the protein. For
example, most
buried amino acid residues require nonpolar side chains, whereas few features
of surface
side chains are generally conserved. Other such phenotypically silent
substitutions are
described in Bowie, J. U. et al., supra, and the references cited therein.
Typically seen as
conservative substitutions are the replacements, one for another, among the
aliphatic
amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser
and Thr,
exchange of the acidic residues Asp and Glu, substitution between the amide
residues Asn
and Gln, exchange of the basic residues Lys and Arg and replacements among the
aromatic residues Phe, Tyr.
[0159] Thus, antibodies of the present invention may bind fragments,
derivatives or
analogs of the polypeptide of SEQ m N0:3228, or that encoded by the deposited
cDNA
plasmid, such as (i) polypeptides in which one or more of the amino acid
residues are
substituted with a conserved or non-conserved amino acid residue (preferably a
conserved
amino acid residue) and such substituted amino acid residue may or may not be
one
encoded by the genetic code, or (ii) polypeptides in which one or more of the
amino acid
residues includes a substituent group, or (iii) polypeptides in which the
extracellular
domain of the polypeptide is fused with another compound, such as a compound
to
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
increase the half-life of the polypeptide (for example, polyethylene glycol),
or (iv)
polypeptides in which the additional amino acids are fused to the
extracellular domain of
the polypeptide, such as an IgG Fc fusion region peptide or leader or
secretory sequence or
a sequence which is employed for purification of the extracellular domain of
the
polypeptide or a proprotein sequence.
[0160] Antibodies of the present invention may bind fragments, derivatives or
analogs
of the polypeptide of SEQ ID N0:3229, or that encoded by the deposited cDNA
plasmid,
such as (i) polypeptides in which one or more of the amino acid residues are
substituted
with a conserved or non-conserved amino acid residue (preferably a conserved
amino acid
residue) and such substituted amino acid residue may or may not be one encoded
by the
genetic code, or (ii) polypeptides in which one or more of the amino acid
residues includes
a substituent group, or (iii) polypeptides in which the extracellular domain
of the
polypeptide is fused with another compound, such as a compound to increase the
half-life
of the polypeptide (for example, polyethylene glycol), or (iv) polypeptides in
which the
additional amino acids are fused to the extracellular domain of the
polypeptide, such as, a
soluble biologically active fragment of another TNF ligand family member
(e.g., CD40
Ligand), an IgG Fc fusion region peptide or leader or secretory sequence or a
sequence
which is employed for purification of the extracellular domain of the
polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are deemed to be
within the
scope of those skilled in the art from the teachings herein.
[0161] Thus, the antibodies of the invention may bind BLyS polypeptides that
include
one or more amino acid substitutions, deletions or additions, either from
natural mutations
or human manipulation. As indicated, changes are preferably of a minor nature,
such as
conservative amino acid substitutions that do not significantly affect the
folding or activity
of the protein (see Table 13).
86
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
TABLE 13. Conservative Amino Acid Substitutions.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic ~ Leucine
Isoleucine
Valine
Polar ~ Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic ~ Aspartic Acid
I Glutamic Acid
Small Alanine
S Brine
Threonine
Methionine
[0162] In one embodiment of the invention, antibodies of the present invention
bind
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of a
BLyS polypeptide having an amino acid sequence which contains at least one
conservative
' amino acid substitution, but not more than 50 conservative amino acid
substitutions, even
more preferably, not more than 40 conservative amino acid substitutions, still
more
preferably, not more than 30 conservative amino acid substitutions, and still
even more
preferably, not more than 20 conservative amino acid substitutions. In one
embodiment of
the invention, antibodies of the present invention bind polypeptides
comprising, or
alternatively consisting of, the amino acid sequence of a BLyS polypeptide
having an
amino acid sequence which contains at least one conservative amino acid
substitution, but
not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid
substitutions.
[0163] For example, site directed changes at the amino acid level of BLyS can
be
made by replacing a particular amino acid with a conservative substitution.
Antibodies of
the present invention may bind BLyS amino acid sequences containing
conservative
-substitution mutations of the polypeptide of SEQ ID N0:3228 including: M1
replaced
87
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
with A, G, I, L, S, T, or V; DZ replaced with E; D3 replaced with E; S4
replaced with A,
G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6 replaced with
D; R7
replaced with H, or K; E8 replaced with D; Q9 replaced with N; S10 replaced
with A, G, I,
L, T, M, or V; R11 replaced with H, or K; L12 replaced with A, G, I, S, T, M,
or V; T13
replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M, or
V; L16
replaced with A, G, I, S, T, M, or V; K17 replaced with H, or R; K18 replaced
with H, or
R; R19 replaced with H, or K; E20 replaced with D; E21 replaced with D; M22
replaced
with A, G, I, L, S, T, or V; K23 replaced with H, or R; L24 replaced with A,
G, I, S, T, M,
or V; K25 replaced with H, or R; E26 replaced with D; V28 replaced with A, G,
I, L, S, T,
or M; S29 replaced with A, G, I, L, T, M, or V; I30 replaced with A, G, L, S,
T, M, or V;
L31 replaced with A, G, I, S, T, M, or V; R33 replaced with H, or K; K34
replaced with
H, or R; E35 replaced with D; S36 replaced with A, G, I, L, T, M, or V; S38
replaced with
A, G, I, L, T, M, or V; V39 replaced with A, G, I, L, S, T, or M; R40 replaced
with H, or
K; S41 replaced with A, G, I, L, T, M, or V; S42 replaced with A, G, I, L, T,
M, or V; K43
replaced with H, or R; D44 replaced with E; G45 replaced with A, I, L, S, T,
M, or V; K46
replaced with H, or R; L47 replaced with A, G, I, S, T, M, or V; L48 replaced
with A, G,
I, S, T, M, or V; A49 replaced with G, I, L, S, T, M, or V; A50 replaced with
G, I, L, S, T,
M, or V; T51 replaced with A, G, I, L, S, M, or V; L52 replaced with A, G, I,
S, T, M, or
V; L53 replaced with A, G, I, S, T, M, or V; L54 replaced with A, G, I, S, T,
M, or V; A55
replaced with G, I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or
V; L57
replaced with A, G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or
V; L61
replaced with A, G, I, S, T, M, or V; T62 replaced with A, G, I, L, S, M, or
V; V63
replaced with A, G, I, L, S, T, or M; V64 replaced with A, G, I, L, S, T, or
M; S65
replaced with A, G, I, L, T, M, or V; F66 replaced with W, or Y; Y67 replaced
with F, or
W; Q68 replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70 replaced
with G,
I, L, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; L72 replaced
with A, G, I, S,
T, M, or V; Q73 replaced with N; G74 replaced with A, I, L, S, T, M, or V; D75
replaced
with E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G, I, L,
S, T, M, or V;
S78 replaced with A, G, I, L, T, M, or V; L79 replaced with A, G, I, S, T, M,
or V; R80
replaced with H, or K; A81 replaced with G, I, L, S, T, M, or V; E82 replaced
with D; L83
replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85 replaced with
A, I, L, S,
T, M, or V; H86 replaced with K, or R; H87 replaced with K, or R; A88 replaced
with G,
8s
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
I, L, S, T, M, or V; E89 replaced with D; K90 replaced with H, or R; L91
replaced with A,
G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or V; G94 replaced
with A, I, L, S,
T, M, or V; A95 replaced with G, I, L, S, T, M, or V; G96 replaced with A, I,
L, S, T, M,
or V; A97 replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R;
A100 replaced
with G, I, L, S, T, M, or V; 6101 replaced with A, I, L, S, T, M, or V; L102
replaced with
A, G, I, S, T, M, or V; E103 replaced with D; E104 replaced with D; A105
replaced with
G, I, L, S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108
replaced with A,
G, I, L, S, T, or M; T109 replaced with A, G, I, L, S, M, or V; Al 10 replaced
with G, I, L,
S, T, M, or V; Gl l l replaced with A, I, L, S, T, M, or V; L112 replaced with
A, G, I, S, T,
M, or V; K113 replaced with H, or R; I114 replaced with A, G, L, S, T, M, or
V; F115
replaced with W, or Y; E116 replaced with D; A119 replaced with G, I, L, S, T,
M, or V;
6121 replaced with A, I, L, S, T, M, or V; E122 replaced with D; 6123 replaced
with A,
I, L, S, T, M, or V; N124 replaced with Q; S 125 replaced with A, G, I, L, T,
M, or V;
5126 replaced with A, G, I, L, T, M, or V; Q127 replaced with N; N128 replaced
with Q;
5129 replaced with A, G, I, L, T, M, or V; 8130 replaced with H, or K; N131
replaced
with Q; K132 replaced with H, or R; 8133 replaced with H, or K; A134 replaced
with G,
I, L, S, T, M, or V; V135 replaced with A, G, I, L, S, T, or M; Q136 replaced
with N;
6137 replaced with A, I, L, S, T, M, or V; E139 replaced with D; E140 replaced
with D;
T141 replaced with A, G, I, L, S, M, or V; V142 replaced with A, G, I, L, S,
T, or M;
T143 replaced with A, G, I, L, S, M, or V; Q144 replaced with N; D145 replaced
with E;
L147 replaced with A, G, I, S, T, M, or V; Q148 replaced with N; L149 replaced
with A,
G, I, S, T, M, or V; I150 replaced with A, G, L, S, T, M, or V; A151 replaced
with G, I, L,
S, T, M, or V; D152 replaced with E; 5153 replaced with A, G, I, L, T, M, or
V; E154
replaced with D; T155 replaced with A, G, I, L, S, M, or V; T157 replaced with
A, G, I, L,
S, M, or V; I158 replaced with A, G, L, S, T, M, or V; Q159 replaced with N;
K160
replaced with H, or R; 6161 replaced with A, I, L, S, T, M, or V; 5162
replaced with A,
G, I, L, T, M, or V; Y163 replaced with F, or W; T164 replaced with A, G, I,
L, S, M, or
V; F165 replaced with W, or Y; V166 replaced with A, G, I, L, S, T, or M; W16$
replaced
with F, or Y; L169 replaced with A, G, I, S, T, M, or V; L170 replaced with A,
G, I, S, T,
M, or V; 5171 replaced with A, G, I, L, T, M, or V; F172 replaced with W, or
Y; K173
replaced with H, or R; 8174 replaced with H, or K; 6175 replaced with A, I, L,
S, T, M,
or V; 5176 replaced with A, G, I, L, T, M, or V; A177 replaced with G, I, L,
S, T, M, or
89
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WO 03/055979 PCT/US02/36496
V; L178 replaced with A, G, I, S, T, M, or V; E179 replaced with D; E180
replaced with
D; K181 replaced with H, or R; E182 replaced with D; N183 replaced with Q;
K184
replaced with H, or R; I185 replaced with A, G, L, S, T, M, or V; L186
replaced with A,
G, I, S, T, M, or V; V 187 replaced with A, G, I, L, S, T, or M; K188 replaced
with H, or
R; E189 replaced with D; T190 replaced with A, G, I, L, S, M, or V; 6191
replaced with
A, I, L, S, T, M, or V; Y192 replaced with F, or W; F193 replaced with W, or
Y; F194
replaced with W, or Y; I195 replaced with A, G, L, S, T, M, or V; Y196
replaced with F,
or W; 6197 replaced with A, I, L, S, T, M, or V; Q198 replaced with N; V199
replaced
with A, G, I, L, S, T, or M; L200 replaced with A, G, I, S, T, M, or V; Y201
replaced with
F, or W; T202 replaced with A, G, I, L, S, M, or V; D203 replaced with E; K204
replaced
with H, or R; T205 replaced with A, G, I, L, S, M, or V; Y206 replaced with F,
or W;
A207 replaced with G, I, L, S, T, M, or V; M208 replaced with A, G, I, L, S,
T, or V;
6209 replaced with A, I, L, S, T, M, or V; H210 replaced with K, or R; L211
replaced
with A, G, I, S, T, M, or V; I212 replaced with A, G, L, S, T, M, or V; Q213
replaced with
N; 8214 replaced with H, or K; K215 replaced with H, or R; K216 replaced with
H, or R;
V217 replaced with A, G, I, L, S, T, or M; I3218 replaced with K, or R; V219
replaced
with A, G, I, L, S, T, or M; F220 replaced with W, or Y; 6221 replaced with A,
I, L, S, T,
M, or V; D222 replaced with E; E223 replaced with D; L224 replaced with A, G,
I, S, T,
M, or V; 5225 replaced with A, G, I, L, T, M, or V; L226 replaced with A, G,
I, S, T, M,
or V; V227 replaced with A, G, I, L, S, T, or M; T228 replaced with A, G, I,
L, S, M, or
V; L229 replaced with A, G, I, S, T, M, or V; F230 replaced with W, or Y; 8231
replaced
with H, or K; I233 replaced with A, G, L, S, T, M, or V; Q234 replaced with N;
N235
replaced with Q; M236 replaced with A, G, I, L, S, T, or V; E238 replaced with
D; T239
replaced with A, G, I, L, S, M, or V; L240 replaced with A, G, I, S, T, M, or
V; N242
replaced with Q; N243 replaced with Q; 5244 replaced with A, G, I, L, T, M, or
V; Y246
replaced with F, or W; 5247 replaced with A, G, I, L, T, M, or V; A248
replaced with G,
I, L, S, T, M, or V; 6249 replaced with A, I, L, S, T, M, or V; I250 replaced
with A, G, L,
S, T, M, or V; A251 replaced with G, I, L, S, T, M, or V; K252 replaced with
H, or R;
L253 replaced with A, G, I, S, T, M, or V; E254 replaced with D; E255 replaced
with D;
6256 replaced with A, I, L, S, T, M, or V; D257 replaced with E; E258 replaced
with D;
L259 replaced with A, G, I, S, T, M, or V; Q260 replaced with N; L261 replaced
with A,
G, I, S, T, M, or V; A262 replaced with G, I, L, S, T, M, or V; I263 replaced
with A, G, L,
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
S, T, M, or V; 8265 replaced with H, or K; E266 replaced with D; N267 replaced
with Q;
A268 replaced with G, I, L, S, T, M, or V; Q269 replaced with N; I270 replaced
with A,
G, L, S, T, M, or V; 5271 replaced with A, G, I, L, T, M, or V; L272 replaced
with A, G,
I, S, T, M, or V; D273 replaced with E; 6274 replaced with A, I, L, S, T, M,
or V; D275
replaced with E; V276 replaced with A, G, I, L, S, T, or M; T277 replaced with
A, G, I, L,
S, M, or V; F278 replaced with W, or Y; F279 replaced with W, or Y; 6280
replaced with
A, I, L, S, T, M, or V; A281 replaced with G, I, L, S, T, M, or V; L282
replaced with A,
G, I, S, T, M, or V; K283 replaced with H, or R; L284 replaced with A, G, I,
S, T, M, or
V; and/or L285 replaced with A, G, I, S, T, M, or V.
[0164] In another embodiment, site directed changes at the amino acid level of
BLyS
can be made by replacing a particular amino acid with a conservative
substitution.
Antibodies of the present invention may bind BLyS amino acid sequences
containing
conservative substitution mutations of the polypeptide of SEQ ID N0:3229
including: M1
replaced with A, G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E;
S4 replaced
with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6
replaced with D;
R7 replaced with H, or K; E8 replaced with D; Q9 replaced with N; S 10
replaced with A,
G, I, L, T, M, or V; R11 replaced with H, or K; L12 replaced with A, G, I, S,
T, M, or V;
T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M,
or V; L16
replaced with A, G, I, S, T, M, or V; K17 replaced with H, or R; K18 replaced
with H, or
R; R19 replaced with H, or K; E20 replaced with D; E21 replaced with D; M22
replaced
with A, G, I, L, S, T, or V; K23 replaced with H, or R; L24 replaced with A,
G, I, S, T, M,
or V; K25 replaced with H, or R; E26 replaced with D; V28 replaced with A, G,
I, L, S, T,
or M; S29 replaced with A, G, I, L, T, M, or V; I30 replaced with A, G, L, S,
T, M, or V;
L31 replaced with A, G, I, S, T, M, or V; R33 replaced with H, or K; K34
replaced with
H, or R; E35 replaced with D; S36 replaced with A, G, I, L, T, M, or V; S38
replaced with
A, G, I, L, T, M, or V; V39 replaced with A, G, I, L, S, T, or M; R40 replaced
with H, or
K; S41 replaced with A, G, I, L, T, M, or V; S42 replaced with A, G, I, L, T,
M, or V; K43
replaced with H, or R; D44 replaced with E; G45 replaced with A, I, L, S, T,
M, or V; K46
replaced with H, or R; L47 replaced with A, G, I, S, T, M, or V; L48 replaced
with A, G,
I, S, T, M, or V; A49 replaced with G, I, L, S, T, M, or V; A50 replaced with
G, I, L, S, T,
M, or V; T51 replaced with A, G, I, L, S, M, or V; L52 replaced with A, G, I,
S, T, M, or
V; L53 replaced with A, G, I, S, T, M, or V; L54 replaced with A, G, I, S, T,
M, or V; A55
91
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replaced with G, I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or
V; L57
replaced with A, G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or
V; L61
replaced with A, G, I, S, T, M, or V; T62 replaced with A, G, I, L, S, M, or
V; V63
replaced with A, G, I, L, S, T, or M; V64 replaced with A, G, I, L, S, T, or
M; S65
replaced with A, G, I, L, T, M, or V; F66 replaced with W, or Y; Y67 replaced
with F, or
W; Q68 replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70 replaced
with G,
I, L, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; L72 replaced
with A, G, I, S,
T, M, or V; Q73 replaced with N; G74 replaced with A, I, L, S, T, M, or V; D75
replaced
with E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G, I, L,
S, T, M, or V;
S78 replaced with A, G, I, L, T, M, or V; L79 replaced with A, G, I, S, T, M,
or V; R80
replaced with H, or K; A81 replaced with G, I, L, S, T, M, or V; E82 replaced
with D; L83
replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85 replaced with
A, I, L, S,
T, M, or V; H86 replaced with K, or R; H87 replaced with K, or R; A88 replaced
with G,
I, L, S, T, M, or V; E89 replaced with D; K90 replaced with H, or R; L91
replaced with A,
G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or V; G94 replaced
with A, I, L, S,
T, M, or V; A95 replaced with G, I, L, S, T, M, or V; G96 replaced with A, I,
L, S, T, M,
or V; A97 replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R;
A100 replaced
with G, I, L, S, T, M, or V; 6101 replaced with A, I, L, S, T, M, or V; L102
replaced with
A, G, I, S, T, M, or V; E103 replaced with D; E104 replaced with D; A105
replaced with
G, I, L, S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108
replaced with A,
G, I, L, S, T, or M; T109 replaced with A, G, I, L, S, M, or V; A110 replaced
with G, I, L,
S, T, M, or V; 6111 replaced with A, I, L, S, T, M, or V; L112 replaced with
A, G, I, S, T,
M, or V; K113 replaced with H, or R; I114 replaced with A, G, L, S, T, M, or
V; F115
replaced with W, or Y; E116 replaced with D; A119 replaced with G, I, L, S, T,
M, or V;
6121 replaced with A, I, L, S, T, M, or V; E122 replaced with D; 6123 replaced
with A,
I, L, S, T, M, or V; N124 replaced with Q; 5125 replaced with A, G, I, L, T,
M, or V;
5126 replaced with A, G, I, L, T, M, or V; Q127 replaced with N; N128 replaced
with Q;
S129 replaced with A, G, I, L, T, M, or V; 8130 replaced with H, or K; N131
replaced
with Q; K132 replaced with H, or R; 8133 replaced with H, or K; A134 replaced
with G,
I, L, S; T, M, or V; V135 replaced with A, G, I, L, S, T, or M; Q136 replaced
with N;
6137 replaced with A, I, L, S, T, M, or V; E139 replaced with D; E140 replaced
with D;
T141 replaced with A, G, I, L, S, M, or V; 6142 replaced with A, I, L, S, T,
M, or V;
92
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
5143 replaced with A, G, I, L, T, M, or V; Y144 replaced with F, or W; T145
replaced
with A, G, I, L, S, M, or V; F146 replaced with W, or Y; V147 replaced with A,
G, I, L, S,
T, or M; W149 replaced with F, or Y; L150 replaced with A, G, I, S, T, M, or
V; L151
replaced with A, G, I, S, T, M, or V; 5152 replaced with A, G, I, L, T, M, or
V; F153
replaced with W, or Y; K154 replaced with H, or R; 8155 replaced with H, or K;
6156
replaced with A, I, L, S, T, M, or V; 5157 replaced with A, G, I, L, T, M, or
V; A158
replaced with G, I, L, S, T, M, or V; L159 replaced with A, G, I, S, T, M, or
V; E160
replaced with D; E161 replaced with D; K162 replaced with H, or R; E163
replaced with
D; N164 replaced with Q; K165 replaced with H, or R; I166 replaced with A, G,
L, S, T,
M, or V; L167 replaced with A, G, I, S, T, M, or V; V168 replaced with A, G,
I, L, S, T,
or M; K169 replaced with H, or R; E170 replaced with D; T171 replaced with A,
G, I, L,
S, M, or V; 6172 replaced with A, I, L, S, T, M, or V; Y173 replaced with F,
or W; F174
replaced with W, or Y; F175 replaced with W, or Y; I176 replaced with A, G, L,
S, T, M,
or V; Y177 replaced with F, or W; 6178 replaced with A, I, L, S, T, M, or V;
Q179
replaced with N; V180 replaced with A, G, I, L, S, T, or M; L181 replaced with
A, G, I, S,
T, M, or V; Y182 replaced with F, or W; T183 replaced with A, G, I, L, S, M,
or V; D184
replaced with E; K185 replaced with H, or R; T186 replaced with A, G, I, L, S,
M, or V;
Y187 replaced with F, or W; A188 replaced with G, I, L, S, T, M, or V; M189
replaced
with A, G, I, L, S, T, or V; 6190 replaced with A, I, L, S, T, M, or V; H191
replaced with
K, or R; L192 replaced with A, G, I, S, T, M, or V; I193 replaced with A, G,
L, S, T, M, or
V; Q194 replaced with N; 8195 replaced with H, or K; K196 replaced with H, or
R; K197
replaced with H, or R; V198 replaced with A, G, I, L, S, T, or M; H199
replaced with K,
or R; V200 replaced with A, G, I, L, S, T, or M; F201 replaced with W, or Y;
6202
replaced with A, I, L, S, T, M, or V; D203 replaced with E; E204 replaced with
D; L205
replaced with A, G, I, S, T, M, or V; 5206 replaced with A, G, I, L, T, M, or
V; L207
replaced with A, G, I, S, T, M, or V; V208 replaced with A, G, I, L, S, T, or
M; T209
replaced with A, G, I, L, S, M, or V; L210 replaced with A, G, I, S, T, M, or
V; F211
replaced with W, or Y; 8212 replaced with H, or K; I214 replaced with A, G, L,
S, T, M,
or V; Q215 replaced with N; N216 replaced with Q; M217 replaced with A, G, I,
L, S, T,
or V; E219 replaced with D; T220 replaced with A, G, I, L, S, M, or V; L221
replaced
with A, G, I, S, T, M, or V; N223 replaced with Q; N224 replaced with Q; 5225
replaced
with A, G, I, L, T, M, or V; Y227 replaced with F, or W; S228 replaced with A,
G, I, L, T,
93
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
M, or V; A229 replaced with G, I, L, S, T, M, or V; 6230 replaced with A, I,
L, S, T, M,
or V; I231 replaced with A, G, L, S, T, M, or V; A232 replaced with G, I, L,
S, T, M, or
V; K233 replaced with H, or R; L234 replaced with A, G, I, S, T, M, or V; E235
replaced
with D; E236 replaced with D; 6237 replaced with A, I, L, S, T, M, or V; D238
replaced
with E; E239 replaced with D; L240 replaced with A, G, I, S, T, M, or V; Q241
replaced
with N; L242 replaced with A, G, I, S, T, M, or V; A243 replaced with G, I, L,
S, T, M, or
V; I244 replaced with A, G, L, S, T, M, or V; 8246 replaced with H, or K; E247
replaced
with D; N248 replaced with Q; A249 replaced with G, I, L, S, T, M, or V; Q250
replaced
with N; I251 replaced with A, G, L, S, T, M, or V; 5252 replaced with A, G, I,
L, T, M, or
V; L253 replaced with A, G, I, S, T, M, or V; D254 replaced with E; 6255
replaced with
A, I, L, S, T, M, or V; D256 replaced with E; V257 replaced with A, G, I, L,
S, T, or M;
T258 replaced with A, G, I, L, S, M, or V; F259 replaced with W, or Y; F260
replaced
with W, or Y; 6261 replaced with A, I, L, S, T, M, or V; A262 replaced with G,
I, L, S, T,
M, or V; L263 replaced with A, G, I, S, T, M, or V; K264 replaced with H, or
R; L265
replaced with A, G, I, S, T, M, or V; and/or L266 replaced with A, G, I, S, T,
M, or V.
[0165] In another embodiment, site directed changes at the amino acid level of
BLyS
can be made by replacing a particular amino acid with a conservative
substitution.
Antibodies of the present invention may bind BLyS amino acid sequences
containing
conservative substitution mutations of the polypeptide of any one of SEQ ID
NOS:3230-
3237.
[0166] Amino acids in the BLyS polypeptides that are essential for function
can be
identified by methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085
(1989)).
The latter procedure introduces single alanine mutations at every residue in
the molecule.
The resulting mutant molecules are then tested for functional activity, such
ligand binding
and the ability to stimulate lymphocyte (e.g., B cell) as, for example,
proliferation,
differentiation, and/or activation. Accordingly, antibodies of the present
invention may
bind amino acids in the BLyS polypeptides that are essential for function. In
preferred
embodiments, antibodies of the present invention bind amino acids in the BLyS
polypeptides that are essential for function and inhibit BLyS polypeptide
function. In
other preferred embodiments, antibodies of the present invention bind amino
acids in the
BLyS polypeptides that are essential for function and enhance BLyS polypeptide
function.
94
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
[0167] Of special interest are substitutions of charged amino acids with other
charged
or neutral amino acids which may produce proteins with highly desirable
improved
characteristics, such as less aggregation. Aggregation may not only reduce
activity but
also be problematic when preparing pharmaceutical formulations, because
aggregates can
be immunogenic (Pinckard et al., Cli>z. Exp. Immu>zol. 2:331-340 (1967);
Robbins et al.,
Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug
Carrier Systems
10:307-377 (1993).
[0168] In another embodiment, the invention provides for antibodies that bind
polypeptides having amino acid sequences containing non-conservative
substitutions of
the amino acid sequence provided in SEQ ID N0:3228. For example, non-
conservative
substitutions of the BLyS protein sequence provided in SEQ ID N0:3228 include:
M1
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q,
F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T5
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L,
S, T, M, V, N,
Q, F, W, Y, P, or C; R7 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C;
ES replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q9
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S 10 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; R11 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; L12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T13 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S 14 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
C15 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
L16 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; K17 replaced with D, E, A, G, I,
L, S, T, M, V,
N, Q, F, W, Y, P, or C; K18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; R19 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E20 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E21 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M22 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; K23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; L24
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; E26 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; C27 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
or P; V28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S29 replaced
with D, E, H,
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
K, R, N, Q, F, W, Y, P, or C; I30 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L31
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R33 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; K34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; E35 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or~
C; S36
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P37 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S38 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; V39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R40 replaced
with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S41 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; S42 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K43
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D44 replaced with H, K,
R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G45 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; K46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L47
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; A49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A50
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T51 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; L52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A55
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L56 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S58
replaced with D, E, H, K, R, N, Q, .F, W, Y, P, or C; C59 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C60 replaced with D, E, H, K, R, A,
G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; T62
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; V64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S65
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F66 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; Y67 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T,
M, V, P, or C; Q68 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C;
V69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A70 replaced with D,
E, H, K,
R, N, Q, F, W, Y, P, or C; A71 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L72
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q73 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; G74 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
96
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
or C; D75 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; L76
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A77 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; S78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L79
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R80 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; A81 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
E82 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L83
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q84 replaced with D, E, H, K, R,
A, G, I, L, S,
T, M, V, F, W, Y, P, or C; G85 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; H86
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H87
replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C;
K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L91
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, or C; A93 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G94
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A95 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A97
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P98 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K99 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; A100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; 6101
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L102 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; E103 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; A105
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P106 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A107 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; V108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T109
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A110 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; 6111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L112 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; K113 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; I114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F115
replaced with
D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E116 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, or C; P118 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q,
97
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
F, W, Y, or C; A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P120
replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 6121 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; E122 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; 6123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N124
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S 125
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; S 126 replaced with D, E, H, K, R, N,
Q, F, W, Y, P,
or C; Q127 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; N128
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S 129
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; 8130 replaced with D, E, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; N131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P,
or C; K132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
8133
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A134
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V135 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; Q136 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;
6137
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E139 replaced with H, K, R, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; E140 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V142
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; T143 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; Q144 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; D145
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 0146
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L147 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; Q148 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W,
Y, P, or C; L149 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I150
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; A151 replaced with D, E, H, K, R, N, Q,
F, W, Y,
P, or C; D152 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; 5153
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E154 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; T155 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; P156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; T157
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I158 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; Q159 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P,
or C; K160 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
6161
98
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WO 03/055979 PCT/US02/36496
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 162 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; Y163 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or
C; T164 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F165 replaced
with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V 166 replaced with D, E, H, K,
R, N, Q, F, W,
Y, P, or C; P167 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C;
W168 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L169
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L170 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; S 171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F172
replaced with
D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K173 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; 8174 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; 6175 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
5176 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A177 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; L178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E179
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E180 replaced with H,
K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K181 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; E182 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; N183 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,
or C; K184
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I185
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L186 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; V187 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K188 replaced
with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E189 replaced with H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; T190 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
6191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y192 replaced with
D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; F193 replaced with D, E, H, K, R, N,
Q, A, G, I,
L, S, T, M, V, P, or C; F194 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P,
or C; I195 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y196 replaced
with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 6197 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; Q198 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C;
V 199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L200 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; Y201 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V,
P, or C; T202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D203
replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K204 replaced with D, E,
A, G, I, L,
99
CA 02467521 2004-05-14
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S, T, M, V, N, Q, F, W, Y, P, or C; T205 replaced with D, E, H, K, R, N, Q, F,
W, Y; P, or
C; Y206 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
A207 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; M208 replaced with D, E, H, K, R,
N, Q, F,
W, Y, P, or C; 6209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Ii210
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L211 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; I212 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Q213
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; 8214
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K215 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; K216 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; V217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H218
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V219
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F220 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S,
T, M, V, P, or C; 6221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D222
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E223
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L224 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; 5225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L226
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V227 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; T228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L229
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F230 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; 8231 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; C232 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
or P; I233 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q234 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N235 replaced with D, E, H,
K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; M236 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; P237 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; E238
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T239
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; L240 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; P241 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; N242
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N243
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; 5244 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; C245 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F,
W, Y, or P; Y246 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; 5247
l00
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A248 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; 6249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I250
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A251 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; K252 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; L253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E254 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E255 replaced with H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; 6256 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; D257 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E258
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L259
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; Q260 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, F, W, Y, P, or C; L261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A262
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I263 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; P264 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or C; 8265 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; E266
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N267
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A268 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; Q269 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y,
P, or C; I270 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5271
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L272 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; D273 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
6274
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D275 replaced with H, K,
R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; V276 replaced with D, E, H, K, R, N, Q,
F, W, Y,
P, or C; T277 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F278
replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F279 replaced with D, E, H,
K, R, N, Q,
A, G, I, L, S, T, M, V, P; or C; 6280 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
A281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L282 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; K283 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; L284 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; andlor L285
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C.
[0169] In an additional embodiment, antibodies of the present invention bind
BLyS
polypeptides comprising, or alternatively consisting of, a BLyS amino acid
sequence in
which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30
and 50) is
101
CA 02467521 2004-05-14
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replaced with the substituted amino acids as described above (either
conservative or
nonconservative).
[0170] In another embodiment of the invention, antibodies of the present
invention
bind BLyS polypeptides with non-conservative substitutions of the sequence
provided in
SEQ ID N0:3229 including: Ml replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D2
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3
replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R7 replaced with D,
E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; E8 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q,
F, W, Y, P, or C; Q9 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C;
S10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R11 replaced with D,
E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; L12 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; T13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S14 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; C 15 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, or P; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K17
replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K18 replaced with
D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; R19 replaced with D, E, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; E20 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; E21 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; M22
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K23 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L24 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
K25 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E26
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C27 replaced with D,
E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V28 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; S29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I30
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L31 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; P32 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; R33
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K34
replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E35 replaced with H, K, R, A,
G, I, L, S, T,
\ M, V, N, Q, F, W, Y, P, or C; S36 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
P37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
S38 replaced
102
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V39 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; R40 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; S41
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S42 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; K43 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
G45
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K46 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; LA~7 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
I~1.8 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A49 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T51
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L52 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L54
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A55 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L57
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S58 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; C59 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, or P; C60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or P; L61
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T62 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V64
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S65 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; F66 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C;
Y67 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q68
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V69 replaced with D,
E, H, K, R,
N, Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A71
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; Q73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P,
or C; G74 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D75 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L76 replaced with D, E, H, K,
R, N, Q, F,
W, Y, P, or C; A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S78
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; L79 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
A81 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E82 replaced with H, K, R, A, G,
I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; L83 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Q84
103
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G85
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; H86 replaced with D, E, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; H87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replaced with
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K90 replaced with D, E, A, G, I,
L, S, T, M, V,
N, Q, F, W, Y, P, or C; L91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P92
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A93
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G94 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G96
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A97 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; P98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; K99
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A100
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; 6101 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; L102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E103 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E104 replaced with H, K, R, A,
G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; A105 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; P106 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; A107
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V 108 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; T109 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A110
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6111 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K113
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I114
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F115 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S,
T, M, V, P, or C; E116 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
C; P117 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; P118
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A119
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P120 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; 6121 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or
C; E122 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
6123
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N124 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; S 125 replaced with D, E, H, K, R, N,
Q, F, W, Y, P,
or C; 5126 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q127 replaced
with D, E,
104
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N128 replaced with D, E, H,
K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; S 129 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or ,
C; 8130 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
N131 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K132 replaced
with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8133 replaced with D, E, A, G, I,
L, S, T, M,
V, N, Q, F, W, Y, P, or C; A134 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C;
V135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q136 replaced with
D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; 6137 replaced with D, E, H, K, R,
N, Q, F, W,
Y, P, or C; P138 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C;
E139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E140 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T141 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; 6142 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C;
5143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y144 replaced with
D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; T145 replaced with D, E, H, K, R, N,
Q, F, W, Y,
P, or C; F146 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or
C; V147
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P148 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W 149 replaced with D, E, H, K, R,
N, Q, A, G, I,
L, S, T, M, V, P, or C; L150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L151
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 152 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; F153 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or
C; K154 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
8155 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6156 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; 5157 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A158
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L159 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; E160 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; E161 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; K162
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E163
replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N164 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; K165 replaced with,D, E, A, G, I, L, S,
T, M, V, N,
Q, F, W, Y, P, or C; I166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L167
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V168 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; K169 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or
105
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
C; E170 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
T171
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6172 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; Y173 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or
C; F174 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
F175 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I176 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; Y177 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P,
or C; 6178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q179 replaced
with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V 180 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; L181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Y182 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T183 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; D184 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; K185 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; T186
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y187 replaced with D, E,
H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; A188 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; M189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6190 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; H191 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; L192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I193
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; Q194 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, F, W, Y, P, or C; 8195 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P,
or C; K196 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K197
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V198
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; H199 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; V200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F201 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 6202 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; D203 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W,
Y, P, or C; E204 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C;
L205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5206 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; L207 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V208
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T209 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; L210 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F211
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 8212
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C213 replaced with D, E, H,
K, R, A, G,
106
CA 02467521 2004-05-14
WO 03/055979 PCT/US02/36496
I, L, S, T, M, V, N, Q, F, W, y, or P; I214 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; Q215 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; N216
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M217
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P218 replaced with D, E, H, K, R, A, G,
I, L, S, T,
M, V, N, Q, F, W, Y, or C; E219 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F,
W, Y, P, or C; T220 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L221
replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P222 replaced with D, E, H, K, R,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; N223 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V,
F, W, Y, P, or C; N224 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or
C; S225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C226 replaced
with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y227 replaced with D, E, H,
K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; 5228 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
A229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 6230 replaced with
D, E, H, K,
R, N, Q, F, W, Y, P, or C; I231 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A232
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K233 replaced with D, E,
A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; L234 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; E235 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E236
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6237
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; D238 replaced with H, K, R, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; E239 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; L240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q241
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L242 replaced with D, E,
H, K, R, N,
Q, F, W, Y, P, or C; A243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I244
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P245 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; 8246 replaced with D, E, A, G, I, L,
S, T, M, V,
N, Q, F, W, Y, P, or C; E247 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y,
P, or C; N248 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,
or C; A249
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q250 replaced with D, E,
H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; I251 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; 5252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L253 replaced
with D, E,
H, K, R, N, Q, F, W, Y, P, or C; D254 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q,
F, W, Y, P, or C; 6255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D256
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replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V257
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; T258 replaced with D, E, H, K, R, N, Q,
F, W, Y, P,
or C; F259 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
F260
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 6261
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A262 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; L263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K264 replaced
with D, E, A,
G, I,~ L, 5, T, M, V, N, Q, F, W, Y, P, or C; L265 replaced with D, E, H, K,
R, N, Q, F, W,
Y, P, or C; and/or L266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
[0171] In another embodiment, site directed changes at the amino acid level of
BLyS
can be made by replacing a particular amino acid with a non-conservative
substitution.
Antibodies of the present invention may bind BLyS amino acid sequences
containing non-
conservative substitution mutations of the polypeptide of any one of SEQ ID
NOS:3230-
3237.
[0172] In an additional embodiment, antibodies of the present invention bind
BLyS
polypeptides comprising, or alternatively consisting of, a BLyS amino acid
sequence in
which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30
and 50) is
replaced with the substituted amino acids as described above (either
conservative or
nonconservative).
[0173] Replacement of amino acids can also change the selectivity of the
binding of a
ligand to cell surface receptors. For example, Ostade et al., Nature 361:266-
268 (1993)
describes certain mutations resulting in selective binding of TNF-alpha to
only one of the
two known types of TNF receptors. Since BLyS is a member of the TNF
polypeptide
family, mutations similar to those in TNF-alpha are likely to have similar
effects in BLyS
polypeptides.
[0174] Sites that are critical for ligand-receptor binding can also be
determined by
structural analysis such as crystallization, nuclear magnetic resonance or
photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al.
Science
255:306-312 (1992)).
[0175] Since BLyS is a member of the TNF-related protein family, mutations may
be
made in sequences encoding amino acids in the TNF conserved domain, e.g., in
positions
Gly-191 through Leu-284 of SEQ ID NO:3228 or in positions Gly-172 through Leu-
265
of SEQ m NO:3229, may modulate rather than completely eliminate functional
activities
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(e.g., biological activities) of BLyS polypeptides or fragments or variants
thereof.
Accordingly, antibodies of the present invention may bind BLyS polypeptides
that have
mutations in the TNF conserved domain. In preferred embodiments, antibodies of
the
present invention may bind BLyS polypeptides that have mutations in the TNF
conserved
domain and act as antagonists of BLyS. In other preferred embodiments,
antibodies of the
present invention may bind BLyS polypeptides that have mutations in the TNF
conserved
domain and act as agonists of BLyS.
[0176] Recombinant DNA technology known to those skilled in the art (see, for
instance, DNA shuffling supra) can be used to create novel mutant proteins or
muteins
including single or multiple amino acid substitutions, deletions, additions or
fusion
proteins. Such modified polypeptides can show, e.g., enhanced activity or
increased
stability. In addition, they may be purified in higher yields and show better
solubility than
the corresponding natural polypeptide, at least under certain purification and
storage
conditions.
[0177] Thus, the invention also encompasses antibodies that bind BLyS
derivatives
and analogs that have one or more amino acid residues deleted, added, or
substituted to
generate BLyS polypeptides, e.g., that are better suited for expression, scale
up, etc., in the
host cells. For example, cysteine residues can be deleted or substituted with
another
amino acid residue in order to eliminate disulfide bridges; N-linked
glycosylation sites can
be altered or eliminated to achieve, for example, expression of a homogeneous
product
that is more easily recovered and purified from yeast hosts which are known
~to
hyperglycosylate N-linked sites. To this end, a variety of amino acid
substitutions at one
or both of the first or third amino acid positions on any one or more of the
glycosylation
recognition sequences in the BLyS polypeptides of the invention, andlor an
amino acid
deletion at the second position of any one or more such recognition sequences
will prevent
glycosylation of the BLyS at the modified tripeptide sequence (see, e.g.,
Miyajimo et al.,
EMBO J 5(6):1193-1197). By way of non-limiting example, mutation of the serine
at
position 244 to alanine either singly or in combination with mutation of the
asparagine at
position 242 to glutamine abolishes glycosylation of the mature soluble form
of BLyS
(e.g., amino acids 134-285 of SEQ m N0:3228) when expressed in the yeast
Piclzea
pastoris. A mutant BLyS polypeptide in which only the asparagine at position
242 is
mutated to glutamine, is still gycosylated when expressed in Pichea pastoris.
In this
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mutant, the glycosylation event may be due to the activation or unmasking of
an O-linked
glyscosylation site at serine 244. Similar mutations affecting glycosylation
could also be
made in the BLyS polypeptide of SEQ ID N0:3229, i.e., aspargine-223 to
glutamine
and/or serine-224 to alanine of SEQ ID NO:3229. Additionally, one or more of
the amino
acid residues of the polypeptides of the invention (e.g., arginine and lysine
residues) may
be deleted or substituted with another residue to eliminate undesired
processing by
proteases such as, for example, furins or kexins. One possible result of such
a mutation is
that BLyS polypeptide of the invention is not cleaved and released from the
cell surface.
Accordingly, antibodies of the invention may bind BLyS derivatives and analogs
that have
one or more amino acid residues deleted, added, or substituted. In other
embodiments,
antibodies of the invention may bind BLyS derivatives, variants or analogs
that are unable
to be cleaved from the cell surface.
[0178] In a specific embodiment, antibodies of the invention bind BLyS
polypeptides
in which Lys-132 andlor Arg-133 of the BLyS sequence shown in SEQ ID N0:3228
is
mutated to another amino acid residue, or deleted altogether, to prevent or
diminish
release of the soluble form of BLyS from cells expressing BLyS. In a more
specific
embodiment, antibodies of the invention bind BLyS polypeptides in which Lys-
132 of the
BLyS sequence shown in SEQ ID N0:3228 is mutated to Ala-132. In another,
nonexclusive specific embodiment, antibodies of the invention bind BLyS
polypeptides in
which Arg-133 of the BLyS sequence shown in SEQ ID N0:3228 is mutated to Ala-
133.
These mutated proteins have uses such as, for example, in ex vivo therapy or
gene therapy,
to engineer cells expressing a BLyS polypeptide that is retained on the
surface of the
engineered cells.
[0179] In a specific embodiment, antibodies of the invention bind BLyS
polypeptides
in which Cys-146 of the BLyS sequence shown in SEQ ID N0:3228 is mutated to
another
amino acid residue, or deleted altogether, for example, to aid preventing or
diminishing
oligomerization of the mutant BLyS polypeptide when expressed in an expression
system.
In a specific embodiment, antibodies of the invention bind BLyS polypeptides
in which
Cys-146 is replaced with a serine amino acid residue.
[0180] In another specific embodiment, antibodies of the invention bind BLyS
polypeptides in which Cys-232 of the BLyS sequence shown in SEQ ID N0:3228 is
mutated to another amino acid residue, or deleted altogether, for example, to
aid
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preventing or diminishing oligomerization of the mutant BLyS polypeptide when
expressed in an expression system. In a specific embodiment, antibodies of the
invention
bind BLyS polypeptides in which Cys-232 is replaced with a serine amino acid
residue.
Polypeptides encoding these polypeptides are also encompassed by the
invention.
[0181] In yet another specific embodiment, antibodies of the invention bind
BLyS
polypeptides in which Cys-245 of the BLyS sequence shown in SEQ ID N0:3228 is
mutated to another amino acid residue, or deleted altogether, for example, to
aid
preventing or diminishing oligomerization of the mutant BLyS polypeptide when
expressed in an expression system. In a specific embodiment, antibodies of the
invention
bind BLyS polypeptides in which Cys-245 is replaced with a serine amino acid
residue.
Polypeptides encoding these polypeptides are also encompassed by the
invention.
[0182] The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purified. A recombinantly produced
version of the
BLyS polypeptides can be substantially purified by the one-step method
described in
Smith and Johnson, Gefie 67:31-40 (1988).
[0183] The antibodies of the present invention bind BLyS polypeptides
including the
complete polypeptide encoded by the deposited cDNA (ATCC Deposit No. 97768)
including the intracellular, transmembrane and extracellular domains of the
polypeptide
encoded by the deposited cDNA, the mature soluble polypeptide encoded by the
deposited
cDNA, the extracellular domain minus the intracellular and transmembrane
domains of the
protein, the complete polypeptide of SEQ ID N0:3228, the mature soluble
polypeptide of
SEQ ID NO:3228, e.g., amino acids 134-285 of SEQ ID N0:3228, the extracellular
domain of SEQ ID N0:3228, amino acid residues 73-285 of SEQ ID N0:3228 minus
the
intracellular and transmembrane domains, as well as polypeptides which have at
least
80%, 85%, 90% similarity, more preferably at least 95% similarity, and still
more
preferably at least 96%, 97%, 98% or 99% similarity to those described above.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
[0184] The antibodies of the present invention bind BLyS polypeptides
including the
complete polypeptide encoded by the deposited cDNA including the
intracellular,
transmembrane and extracellular domains of the polypeptide encoded by the
deposited
cDNA (ATCC Deposit No. 203518), the mature soluble polypeptide encoded by the
deposited cDNA, the extracellular domain minus the intracellular and
transmembrane
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domains of the protein, the complete polypeptide of SEQ m N0:3229, the mature
soluble
of SEQ ID NO:3229, e.g., amino acid residues 134-266 of SEQ m N0:3229, the
extracellular domain of SEQ JD NO:3229, e.g., amino acid residues 73-266 of
SEQ ID
N0:3229 minus the intracellular and transmembrane domains, as well as
polypeptides
which have at least 80%, 85%, 90% similarity, more preferably at least 95%
similarity,
and still more preferably at least 96%, 97%, 98% or 99% similarity to those
described
above. Polynucleotides encoding these polypeptides are also encompassed by the
invention.
[0185] Further antibodies of the present invention bind polypeptides including
polypeptides at least 80%, or at least 85% identical, more preferably at least
90% or 95%
identical, still more preferably at least 96%, 97%, 98% or 99% identical to
the polypeptide
encoded by the deposited cDNA (ATCC Deposit No. 97768) or to the polypeptide
of SEQ
ID N0:3228, and also include antibodies that bind portions of such
polypeptides with at
least 30 amino acids and more preferably at least 50 amino acids.
[0186] Further antibodies of the present invention bind polypeptides including
polypeptides at least 80%, or at least 85% identical, more preferably at least
90% or 95%
identical, still more preferably at least 96%, 97%, 98% or 99% identical to
the. polypeptide
encoded by the deposited cDNA (ATCC Deposit No. 203518) or to the polypeptide
of
SEQ ID N0:3229, and also include antibodies that bind portions of such
polypeptides with
at least 30 amino acids and more preferably at least 50 amino acids.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[0187] By "% similarity" for two polypeptides is intended a similarity score
produced
by comparing the amino acid sequences of the two polypeptides using the
Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group,
University Research Park, 575 Science Drive, Madison, WI 53711) and the
default
settings for determining similarity. Bestfit uses the local homology algorithm
of Smith
and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the
best
segment of similarity between two sequences.
[0188] By a polypeptide having an amino acid sequence at least, for example,
95%
"identical" to a reference amino acid sequence of a BLyS polypeptide is
intended that the
amino acid sequence of the polypeptide is identical to the reference sequence
except that
the polypeptide sequence may include up to five amino acid alterations per
each 100
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amino acids of the reference amino acid of the BLyS polypeptide. In other
words, to
obtain a polypeptide having an amino acid sequence at least 95% identical to a
reference
amino acid sequence, up to 5% of the amino acid residues in the reference
sequence may
be deleted or substituted with another amino acid, or a number of amino acids
up to 5% of
the total amino acid residues in the reference sequence may be inserted into
the reference
sequence. These alterations of the reference sequence may occur at the amino
or carboxy
terminal positions of the reference amino acid sequence or anywhere between
those
terminal positions, interspersed either individually among residues in the
reference
sequence or in one or more contiguous groups within the reference sequence.
[0189] As a practical matter, whether any particular polypeptide is at least
80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid
sequence of
SEQ ID N0:3228, the amino acid sequence encoded by the deposited cDNA clone
HNEDU15 (ATCC Accession No. 97768), or fragments thereof, or, for instance, to
the
amino acid sequence of SEQ ID N0:3229, the amino acid sequence encoded by the
deposited cDNA clone HDPMC52 (ATCC Accession No. 203518), or fragments
thereof,
can be determined conventionally using known computer programs such the
Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer
Group, University Research Park, 575 Science Drive, Madison, WI 53711). When
using
Bestfit or any other sequence alignment program to determine whether a
particular
sequence is, for instance, 95% identical to a reference sequence according to
the present
invention, the parameters are set, of course, such that the percentage of
identity is
calculated over the full length of the reference amino acid sequence and that
gaps in
homology of up to 5% of the total number of amino acid residues in the
reference
sequence are allowed.
[0190] In a specific embodiment, the identity between a reference (query)
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
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According to this embodiment, if the subject sequence is shorter than the
query sequence
due to N- or C-terminal deletions, not because of internal deletions, a manual
correction is
made to the results to take into consideration the fact that the FASTDB
program does not
account for N- and C-terminal truncations of the subject sequence when
calculating global
percent identity. For subject sequences truncated at the N- and C-termini,
relative to the
query sequence, the percent identity is corrected by calculating the number of
residues of
the query sequence that are N- and C-terminal of the subject sequence, which
are not
matched/aligned with a corresponding subject residue, as a percent of the
total bases of the
query sequence. A determination of whether a residue is matched/aligned is
determined
by results of the FASTDB sequence alignment. This percentage is then
subtracted from
the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arrive at a final percent identity score. This final percent
identity score is
what is used for the purposes of this embodiment. Only residues to the N- and
C-termini
of the subject sequence, which are not matched/aligned with the query
sequence, are
considered for the purposes of manually adjusting the percent identity score.
That is, only
query residue positions outside the farthest N- and C-terminal residues of the
subject
sequence. For example, a 90 amino acid residue subject sequence is aligned
with a 100
residue query sequence to determine percent identity. The deletion occurs at
the
N-terminus of the subject sequence and therefore, the FASTDB alignment does
not show a
matchin~alignment of the first 10 residues at the N-terminus. The 10 unpaired
residues
represent 10% of the sequence (number of residues at the N- and C-termini not
matched/total number of residues in the query sequence) so 10% is subtracted
from the
percent identity score calculated by the FASTDB program. If the remaining 90
residues
were perfectly matched the final percent identity would be 90%. In another
example, a 90
residue subject sequence is compared with a 100 residue query sequence. This
time the
deletions are internal deletions so there are no residues at the N- or C-
termini of the
subject sequence which are not matched/aligned with the query. In this case
the percent
identity calculated by FASTDB is not manually corrected. Once again, only
residue
positions outside the N- and C-terminal ends of the subject sequence, as
displayed in the
FASTDB alignment, which are not matched/aligned with the query sequence are
manually
corrected for. No other manual corrections are made for the purposes of this
embodiment.
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Antibodies that Immunospecifically bind BLyS Polypeptides
[0191] The present invention also encompasses antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to BLyS polypeptides, which antibodies comprise, or
alternatively consist of, all or a portion of a heavy and/or light chain
variable domain of
the scFvs referred to in Table 1.
[0192] The present invention also encompasses methods and compositions for
detecting, diagnosing and/or prognosing diseases or disorders associated with
aberrant
BLyS or BLyS receptor expression or inappropriate BLyS or BLyS receptor
function in an
animal, preferably a mammal, and most preferably a human, comprising using
antibodies
(including molecules which comprise, or alternatively consist of, antibody
fragments or
variants thereof) that immunospecifically bind to BLyS. Diseases and disorders
which can
be detected, diagnosed or prognosed with the antibodies of the invention
include, but are
not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple
sclerosis,
myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome),
inflammatory
disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis),
infectious diseases
(e.g., AmS), and proliferative disorders (e.g., leukemia, carcinoma, and
lymphoma).
[0193] The present invention further encompasses methods and compositions for
preventing, treating or ameliorating diseases or disorders associated with
aberrant BLyS or
BLyS receptor expression or inappropriate BLyS or BLyS receptor function in an
animal,
preferably a mammal, and most preferably a human, comprising administering to
said
animal an effective amount of one or more antibodies (including molecules
which
comprise, or alternatively consist of, antibody fragments or variants thereof)
that
immunospecifically bind to BLyS. Diseases and disorders which can be
prevented, treated
or inhibited by administering an effective amount of one or more antibodies or
molecules
of the invention include, but are not limited to, immune disorders (e.g.,
lupus, rheumatoid
arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and
immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic
disorders,
and rheumatoid arthritis), infectious diseases (e.g., AIDS), and proliferative
disorders
(e.g., leukemia, carcinoma, and lymphoma).
Anti-BL~,S Antibodies
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[0194] The antibodies of the present invention were discovered, in part, using
phage
display technology. Single chain antibody molecules ("scFvs") displayed on the
surface
of phage particles were screened to identify those scFvs that
immunospecifically bind to
BLyS, including the membrane-bound form and soluble form of BLyS. The present
invention encompasses the scFvs and portions thereof that were identified to
immunospecifically bind to BLyS, including scFvs that immunospecifically bind
to the
soluble form of BLyS, scFvs that immunospecifically bind to the membrane-bound
form
of BLyS, and scFvs that immunospecifically bind to both the soluble form and
membrane-
bound form of BLyS. In particular, the present invention encompasses scFvs
comprising,
or alternatively consisting of, the amino acid sequence of SEQ ID NOS: 1 -
2128, as
referred to in Table 1. Preferably, the scFvs of the present invention
comprise, or
alternatively consist of, the amino acid sequence of SEQ ID NOS:l - 46, 321 -
329, 834 -
872, 1563 - 1595, or 1881 - 1908. The scFvs include scFvs that bind to soluble
BLyS
(e.g., scFvs comprising, or alternatively consisting of, an amino acid
sequence of SEQ ID
NOS: 1563 - 1880), scFvs that bind to the membrane-bound form of BLyS (e.g.,
scFvs
comprising, or alternatively consisting of, an amino acid sequence of SEQ ID
NOS: 1881 -
2128), and scFvs that bind to both the soluble form and the membrane-bound
form of
BLyS (e.g., scFvs comprising, or alternatively consisting of, an amino acid
sequence of
SEQ ID NOS: 1 - 1562). Molecules comprising, or alternatively consisting of,
fragments
or variants of these scFvs, that immunospecifically bind to BLyS are also
encompassed by
the invention, as are nucleic acid molecules encoding these scFvs, molecules,
fragments
andlor variants.
[0195] In one embodiment of the present invention, scFvs that
immunospecifically
bind to BLyS comprise a polypeptide having the amino acid sequence of any one
of the
VH domains referred to in Table 1 and/or any one of the VL domains referred to
in Table
1. In preferred embodiments, scFvs of the present invention comprise the amino
acid
sequence of a VH domain and VL domain from the same scFv referred to in Table
1. In
alternative embodiments, scFvs of the present invention comprise the amino
acid sequence
of a VH domain and VL domain from different scFvs referred to in Table 1. In
another
embodiment, scFvs that immunospecifically bind to BLyS, comprise a polypeptide
having
the amino acid sequence of any one, two, three, or more of the VH CDRs
referred to in
Table 1 and/or any one, two, three, or more of the VL CDRs referred to in
Table 1. In
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preferred embodiments, scFvs of the present invention comprise the amino acid
sequence
of a VH CDR and VL CDR from the same scFv referred to in Table 1. In
alternative
embodiments, scFvs of the present invention comprise the amino acid sequence
of a VH
CDR and VL CDR from different scFvs referred to in Table 1. Molecules
comprising, or
alternatively consisting of, antibody fragments or variants of the scFvs
referred to in Table
1 that immunospecifically bind to BLyS are also encompassed by the invention,
as are
nucleic acid molecules encoding these scFvs, molecules, fragments and/or
variants.
[0196] (Table 1 can be found at the end of the specification just prior to the
claims.)
[0197] In another embodiment of the present invention, an scFv that
immunospecifically binds to a soluble form of BLyS, comprises, or
alternatively consists
of, the amino acid sequence of SEQ ID NOS:1563 - 1880 as referred to in Table
1. In a
preferred embodiment, an scFv that immunospecifically binds to a soluble form
of BLyS
comprises, or alternatively consists of, the amino acid sequence of SEQ ID
NOS:1570 -
1595. In an even more preferred embodiment, an scFv that immunospecifically
binds to a
soluble form of BLyS comprises, or alternatively consists of, the amino acid
sequence of
SEQ ID NOS:1563 - 1569.
[0198] In another embodiment of the present invention, an scFv that
immunospecifically binds to a membrane-bound form of BLyS comprises, or
alternatively
consists of, the amino acid sequence of SEQ ID NOS:1881 - 2128 as referred to
in Table
1. In a preferred embodiment, an scFv that immunospecifically binds to a
membrane-
bound form of BLyS comprises, or alternatively consists of, the amino acid
sequence of
SEQ ID NOS:1886 - 1908. In an even more preferred embodiment, an scFv that
immunospecifically binds to a membrane-bound form of BLyS comprises, or
alternatively
consists of, the amino acid sequence of SEQ ID NOS:1881 - 1885.
[0199] In another embodiment of the present invention, an scFv that
immunospecifically binds to both the soluble form and membrane-bound form of
BLyS
comprises, or alternatively consists of, the amino acid sequence of SEQ ID
NOS:l - 1562
as referred to in Table 1. In a preferred embodiment, an scFv that
immunospecifically
binds to both the soluble form and membrane-bound form of BLyS comprises, or
alternatively consists of, the amino acid sequence of SEQ ID NOS:834 - 872. In
another
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preferred embodiment, an scFv that immunospecifically binds to both the
soluble form
and membrane-bound form of BLyS comprises, or alternatively consists of, any
one of the
amino acids sequences of SEQ ID NOS:1- 46 or 321 - 329. Molecules comprising,
or alternatively consisting of, fragments or variants of these scFvs, that
immunospecifically bind to the soluble form of BLyS and/or the membrane-bound
form of
BLyS are also encompassed by the invention, as are nucleic acid molecules
encoding these
scFvs, molecules, fragments and/or variants.
[0200] In another embodiment of the present invention, scFvs that
immunospecifically
bind to the soluble form of BLyS, comprise a polypeptide having the amino acid
sequence
of any one of the VH domains contained in SEQ ID NOS:1563 - 1880 as disclosed
in
Table 1 and/or any one of the VL domains contained in SEQ ID NOS:1563 - 1880
as
disclosed in Table 1. In preferred embodiments, scFvs of the present invention
that
immunospecifically bind to the soluble form of BLyS, comprise a polypeptide
having the
amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in
Table
1. In alternative embodiments, scFvs of the present invention that
immunospecifically
bind to the soluble form of BLyS, comprise a polypeptide having amino acid
sequence of
a VH CDR and VL CDR from different scFvs referred to in Table 1. In another
embodiment, scFvs that immunospecifically bind to the soluble form of BLyS,
comprise a
polypeptide having the amino acid sequence of any one, two, three, or more of
the VH
CDRs SEQ DJ NOS:1563 - 1880 as disclosed in Table 1 and/or any one, two,
three, or
more of the VL CDRs contained in contained SEQ ID NOS:1563 - 1880, as
disclosed in
Table 1. In preferred embodiments, scFvs of the present invention that
immunospecifically bind to the soluble form of BLyS, comprise a polypeptide
having the
amino acid sequence of a VH domain and VL domain from the same scFv referred
to in
Table 1. In alternative embodiments, scFvs of the present invention that
immunospecifically bind to the soluble form of BLyS, comprise a polypeptide
having the
of the amino acid sequence of a VH domain and VL domain from different scFvs
referred
to in Table 1. In a preferred embodiment, scFvs that immunospecifically bind
to the
soluble form of BLyS, comprise a polypeptide having the amino acid sequence of
any one
of the VH CDR3s contained in SEQ ID NOS:1563 - 1880 as disclosed in Table 1
and/or
any one of the VL CDR3s contained in SEQ ID NOS: 1563 - 1880 as disclosed in
Table 1.
In preferred embodiments, scFvs of the present invention that
immunospecifically bind to
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the soluble form of BLyS, comprise a polypeptide having the amino acid
sequence of a
VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative
embodiments, scFvs of the present invention that immunospecifically bind to
the soluble
form of BLyS, comprise a polypeptide having the amino acid sequence of a VH
CDR and
VL CDR from different scFvs referred to in Table 1. Molecules comprising, or
alternatively consisting of, fragments or variants of these scFvs, that
immunospecifically
bind to BLyS, preferably the soluble form of BLyS, are also encompassed by the
invention, as are nucleic acid molecules encoding these scFvs, molecules,
fragments
and/or variants.
[0201] In another embodiment of the present invention, scFvs that
immunospecifically
bind to the membrane-bound form of BLyS comprise a polypeptide having the
amino acid
sequence of any one of the VH domains contained in SEQ ID NOS:1881 - 2128 as
disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS:
1881 -
2128 as disclosed in Table 1. In preferred embodiments, scFvs of the present
invention
that immunospecifically bind to the soluble form of BLyS, comprise a
polypeptide having
the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to
in
Table 1. In alternative embodiments, scFvs of the present invention that
immunospecifically bind to the membrane-bound form of BLyS, comprise a
polypeptide
having the amino acid sequence of a VH domain and VL domain from different
scFvs
referred to in Table 1. In another embodiment, scFvs that immunospecifically
bind to the
membrane-bound form of BLyS, comprise a polypeptide having the amino acid
sequence
of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS: 1881 -
2128
as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs
contained in
SEQ ID NOS: 1881 - 2128 as disclosed in Table 1. In preferred embodiments,
scFvs of the
present invention that immunospecifically bind to the membrane-bound form of
BLyS,
comprise a polypeptide having the amino acid sequence of a VH domain and VL
domain
from the same scFv referred to in Table 1. In alternative embodiments, scFvs
of the
present invention that immunospecifically bind to the membrane-bound form of
BLyS,
comprise a polypeptide having the amino acid sequence of a VH domain and VL
domain
from different scFvs referred to in Table 1. In a preferred embodiment, scFvs
that
immunospecifically bind to the membrane-bound form of BLyS, comprise a
polypeptide
having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID
NOS:
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1881- 2128 as disclosed in Table 1 and/or any one of the VL CDR3s contained in
SEQ ID
NOS: 1881 - 2128 as disclosed in Table 1. In preferred embodiments, scFvs of
the present
invention that immunospecifically bind to the membrane-bound form of BLyS,
comprise a
polypeptide having the amino acid sequence of a VH domain and VL domain from
the
same scFv referred to in Table 1. In alternative embodiments, scFvs of the
present
invention that immunospecifically bind to the membrane-bound form of BLyS,
comprise a
polypeptide having the amino acid sequence of a VH CDR and VL CDR from
different
scFvs referred to in Table 1. Molecules comprising, or alternatively
consisting of,
fragments or variants of these scFvs, that immunospecifically bind to BLyS,
preferably the
membrane-bound form of BLyS, are also encompassed by the invention, as are
nucleic
acid molecules encoding these scFvs, molecules, fragments and/or variants.
[0202] In another embodiment of the present invention, scFvs that
immunospecifically
bind to the soluble form and membrane-bound form of BLyS, comprise a
polypeptide
having the amino acid sequence of any one of the VH domains contained in SEQ
ID
NOS:1 - 1562 as disclosed in Table 1 and/or any one of the VL domains
contained in SEQ
ID NOS:1 - 1562 as disclosed in Table 1. In preferred embodiments, scFvs of
the present
invention that immunospecifically bind to the soluble and membrane-bound forms
of
BLyS, comprise a polypeptide having the amino acid sequence of a VH domain and
VL
domain from the same scFv referred to in Table 1. In alternative embodiments,
scFvs of
the present invention that immunospecifically bind to the soluble form and
membrane-
bound form of BLyS, comprise a polypeptide having the amino acid sequence of a
VH
domain and VL domain from different scFvs referred to in Table 1. In another
embodiment, scFvs that immunospecifically bind to the soluble form and
membrane-
bound form of BLyS comprise a polypeptide having the amino acid sequence of
any one,
two, three, or more of the VH CDRs contained in SEQ ID NOS:1 - 1562 as
disclosed in
Table 1 and/or any one, two, three, or more of the VL CDRs contained in SEQ ID
NOS:1
- 1562 as disclosed in Table 1. In preferred embodiments, scFvs of the present
invention
that immunospecifically bind to the soluble form and membrane-bound form of
BLyS,
comprise a polypeptide having the amino acid sequence of a VH domain and VL
domain
from the same scFv referred to in Table 1. In alternative embodiments, scFvs
of the
present invention that immunospecifically bind to the soluble and membrane-
bound forms
of BLyS, comprise a polypeptide having the amino acid sequence of a VH domain
and VL
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domain from different scFvs referred to in Table 1. In a preferred embodiment,
scFvs that
immunospecifically bind to the soluble and membrane-bound forms of BLyS,
comprise a
polypeptide having the amino acid sequence of any one of the VH CDR3s
contained in
SEQ ID NOS:1 - 1562 as disclosed in Table 1 and/or any one of the VL CDR3s
contained
in SEQ ID NOS:1 - 1562, as disclosed in Table 1. In preferred embodiments,
scFvs of the
present invention that immunospecifically bind to the soluble and membrane-
bound forms
of BLyS, comprise a polypeptide having the amino acid sequence of a VH CDR and
VL
CDR from the same scFv referred to in Table 1. In alternative embodiments,
scFvs of the
present invention that immunospecifically bind to the soluble and membrane-
bound forms
of BLyS, comprise a polypeptide having the amino acid sequence of a VH CDR and
VL
CDR from different scFvs referred to in Table 1. Molecules comprising, or
alternatively
consisting of, fragments or variants of these scFvs or molecules, that
immunospecifically
bind to BLyS, preferably the soluble and membrane-bound forms of BLyS, are
also
encompassed by the invention, as are nucleic acid molecules encoding these
scFvs,
molecules, fragments and/or variants.
[0203] The present invention provides antibodies (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof) that
immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS. In
particular, the invention provides antibodies corresponding to the scFvs
referred to in
Table 1, such scFvs may routinely be "converted" to immunoglobulin molecules
by
inserting, for example, the nucleotide sequences encoding the VH and/or VL
domains of
the scFv into an expression vector containing the constant domain sequences
and
engineered to direct the expression of the immunoglobulin molecule, as
described in more
detail in Example 20, infra.
[0204] In one embodiment, the invention provides antibodies (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) wherein
said antibodies comprise, or alternatively consist of, a polypeptide having an
amino acid
sequence of any one of the VH domains contained in the sequences referred to
in Table 1.
The present invention also provides antibodies that immunospecifically bind to
a
polypeptide, or polypeptide fragment of BLyS, wherein said antibodies
comprise, or
alternatively consist of, a polypeptide having an amino acid sequence of any
one, two,
three, or more of the VH CDRs contained in the sequences referred to in Table
1.
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Molecules comprising, or alternatively consisting of, these antibodies, or
antibody
fragments or variants thereof, that immunospecifically bind to BLyS or a BLyS
fragment
are also encompassed by the invention, as are nucleic acid molecules encoding
these
antibodies, molecules, fragments and/or variants.
[0205] In one embodiment of the present invention, antibodies (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind BLyS, comprise, or alternatively consist of, a
polypeptide having
the amino acid sequence of a VH CDR referred to in Table 1. In particular, the
invention
provides antibodies that immunospecifically bind BLyS, comprising, or
alternatively
consisting of, a polypeptide having the amino acid sequence of a VH CDR1
contained in
SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, or 1881 - 1885 as disclosed in
Table 1. In
another embodiment, antibodies that immunospecifically bind BLyS, comprise, or
alternatively consist of, a polypeptide having the amino acid sequence of a VH
CDR2
contained in SEQ D~ NOS:1 - 46, 321 - 329, 1563 - 1569, or 1881- 1885 as
disclosed in
Table 1. In a preferred embodiment, antibodies that immunospecifically bind
BLyS,
comprise, or alternatively consist of a polypeptide having the amino acid
sequence of a
VH CDR3 contained in SEQ ll~ NOS:1 - 46, 321 - 329, 1563 - 1569, or 1881 -
1885 as
disclosed in Table 1. In yet another embodiment, antibodies that
immunospecifically bind
BLyS, comprise, or alternatively consist of, a polypeptide having the amino
acid sequence
of a VH CDR1 contained in SEQ >D NOS:834 - 872, 1570 - 1595, or 1886 - 1908 as
disclosed in Table 1; a VH CDR2 contained in SEQ ID NOS: SEQ ID NOS: SEQ ID
NOS:834 - 872, 1570 - 1595, or 1886 - 1908; and/or a VH CDR3 contained in SEQ
ID
NOS: SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 - 1908 as disclosed in Table
1.
Preferably, antibodies of the invention comprise, or alternatively consist of,
VH CDRs that
are derived from the same scFv as disclosed in Table 1. Molecules comprising,
or
alternatively consisting of, fragments or variants of these anribocues that
immunospecifically bind to BLyS are also encompassed by the invention, as are
nucleic
acid molecules encoding these antibodies, molecules, fragments or variants.
[0206] The present invention provides antibodies (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants) that
immunospecifically bind
to a polypeptide, or polypeptide fragment of BLyS. In particular, the
invention provides
antibodies wherein said antibodies comprise, or alternatively consist of, a VL
domain
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having an amino acid sequence of any one of the VL domains referred to in
Table 1. The
present invention also provides antibodies that immunospecifically bind to a
polypeptide
or polypeptide fragment of BLyS, wherein said antibodies comprise, or
alternatively
consist of, a VL CDR having an amino acid sequence of any one, two, three, or
more of
the VL CDRs contained in the sequences referred to in Table 1. Molecules
comprising, or
alternatively consisting of, fragments or variants of these antibodies that
immunospecifically bind to BLyS are also encompassed by the invention, as are
nucleic
acid molecules encoding these antibodies, molecules, fragments or variants.
[0207] In one embodiment of the present invention, antibodies (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind BLyS, comprise, or alternatively consist of, a
polypeptide having
the amino acid sequence of a VL CDR referred to in Table 1. In particular, the
invention
provides antibodies that immunospecifically bind BLyS, comprising, or
alternatively
consisting of, a polypeptide having the amino acid sequence of a VL CDR1
contained in
SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, or 1881- 1885 as disclosed in Table
1. In
another embodiment, antibodies that immunospecifically bind BLyS comprise, or
alternatively consist of, a polypeptide having the amino acid sequence of a VL
CDR2
contained in SEQ ID NOS:l - 46, 321 - 329, 1563 - 1569, or 1881- 1885 as
disclosed in
Table 1. In a preferred embodiment, antibodies comprise, or alternatively
consist of, a
polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID
NOS: in
SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, or 1881-1885 disclosed in Table 1.
In yet
another embodiment, antibodies that immnospecifically bind BLyS comprise, or
alternatively consist of: a polypeptide having the amino acid sequence of a VL
CDR1
contained in SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 -1908 as disclosed in
Table 1;
a VL CDR2 SEQ ID NOS :834 - 872, 1570 - 1595, or 1886 - 1908 as disclosed in
Table
1; and a VL CDR3 contained SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 - 1908
as
disclosed in Table 1. Preferably, antibodies of the invention comprise, or
alternatively
consist of, VL CDRs that are derived from the same scFv as disclosed in Table
1.
Molecules comprising, or alternatively consisting of, fragments or variants of
these
antibodies, that immunospecifically bind to BLyS are also encompassed by the
invention,
as are nucleic acid molecules encoding these antibodies, molecules, fragments
or variants.
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[0208] The present invention also provides antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS,
wherein
said antibodies comprise, or alternatively consist of, a VH domain of one of
the scFvs
referred to in Table 1 combined with a VL domain of one of the scFvs referred
to in Table
1, or other VL domain. The present invention further provides antibodies
(including
molecules comprise, or alternatively consist of, antibody fragments or
variants thereof)
that immunospecifically bind to a polypeptide or a polypeptide fragment of
BLyS,
wherein said antibodies comprise, or alternatively consist of, a VL domain of
one of the
scFvs referred to in Table 1 combined with a VH domain of one of the scFvs
referred to in
Table 1, or other VH domain. In a preferred embodiment, antibodies that
immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS,
comprise,
or alternatively consist of, a polypeptide having the amino acid sequence of a
VH domain
contained SEQ ID NOS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908
as
disclosed in Table 1 and a VL domain contained in contained SEQ ID NOS:1 - 46,
321 -
329, 834 - 872, 1563 - 1595, or 1881-1908 as disclosed in Table 1. In a
further preferred
embodiment, the antibodies of the invention comprise, or alternatively consist
of, a VH
and a VL domain from the same scFv as disclosed in Table 1. Molecules
comprising, or
alternatively consisting of, fragments or variants of these antibodies, that
immunospecifically bind to BLyS are also encompassed by the invention, as are
nucleic
acid molecules encoding these antibodies, molecules, fragments or variants.
[0209] The present invention also provides antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants)
that
inimunospecifically bind to a polypeptide or polypeptide fragment of BLyS,
wherein said
antibodies comprise, or alternatively consist of, one, two, three, or more VH
CDRs and
one, two, three or more VL CDRs, as referred to in Table 1. In particular, the
invention
provides for antibodies that immunospecifically bind to a polypeptide or
polypeptide
fragment of BLyS, wherein said antibodies comprise, or alternatively consist
of, a VH
CDR1 and a VL CDRl, a VH CDRl and a VL CDR2, a VH CDR1 and a VL CDR3, a
VH CDR2 and a VL CDR1, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a
VH CDR3 and a VH CDR1, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3,
or any combination thereof, of the VH CDRs and VL CDRs referred to in Table 1.
In a
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preferred embodiment, one or more of these combinations are from the same scFv
as
disclosed in Table 1. Molecules comprising, or alternatively consisting of,
fragments or
variants of these antibodies, that immunospecifically bind to BLyS are also
encompassed
by the invention, as are nucleic acid molecules encoding these antibodies,
molecules,
fragments or variants.
[0210] In a preferred embodiment the invention provides antibodies wherein the
VH
CDRX (where X=1, 2, or 3) and VL CDRY (where Y= 1, 2, or 3) are from scFvs
with the
same specificity (i.e., from scFvs that bind soluble BLyS, from scFvs that
bind membrane-
bound BLyS, or from scFvs that bind both soluble and membrane-bound BLyS.
Molecules comprising, or alternatively consisting of, fragments or variants of
these
antibodies, that immunospecifically bind to BLyS are also encompassed by the
invention,
as are nucleic acid molecules encoding these antibodies, molecules, fragments
or variants.
[0211] The term "antibody," as used herein, refers to immunoglobulin molecules
and
immunologically active portions of immunoglobulin molecules, i.e., molecules
that
contain an antigen binding site that immunospecifically binds an antigen. As
such, the
term "antibody" encompasses not only whole antibody molecules, but also
antibody
fragments, as well as variants (including derivatives) of antibodies and
antibody
fragments. Antibodies of the invention include, but are not limited to,
monoclonal,
multispecific, human or chimeric antibodies, single chain antibodies, single
chain Fvs
(scFvs), Fab fragments, F(ab')2 fragments, Fd fragments, disulfide-linked Fvs
(sdFvs),
antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the
invention), and epitope-binding fragments of any of the above. The
immunoglobulin
molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA
and IgY),
class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI and IgA2) or subclass of
imrnunoglobulin
molecule. The antibodies of the present invention also include molecules
comprising, or
alternatively consisting of, a polypeptide having an amino acid sequence of a
portion of
an amino acid sequence contained SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563
- 1595,
or 1881 - 1908. Preferably, an antibody of the invention comprises, or
alternatively
consists of, a polypeptide having an amino acid sequence of a VH domain, VH
CDR, VL
domain, or VL CDR of any one those contained in the sequences referred to in
Table 1.
Antibodies of the invention also include molecules comprising, or
alternatively consisting
of, fragments or variants of the above antibodies that immunospecifically bind
BLyS.
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[0212] Most preferably the antibodies of the present invention are whole
antibodies or
antibody fragments that immunospecifically bind human BLyS. Antibody fragments
of
the invention that immunospecifically bind human BLyS include, but are not
limited to,
Fab, Fab' and F(ab')2, Fd fragments, single-chain Fvs (scFv), single-chain
antibodies,
disulfide-linked Fvs (sdFvs), fragments comprising, or alternatively
consisting of, either a
VL or VH domain, and epitope binding fragments of any of the above.
[0213] BLyS-binding antibody fragments, including single-chain antibodies, may
comprise, or alternatively consist of, the variable regions) alone or in
combination with
the entirety or a portion of the following: hinge region, CHl, CH2, and CH3
domains. In
a preferred embodiment, the antibodies of the invention comprise, or
alternatively consist
of, a polypeptide that immunospecifically binds to BLyS, said polypeptides
comprise, or
alternatively consist of, one, two, three, four, five, six or more CDRs
referred to in Table
1, preferably a polypeptide having an amino acid sequence of a VH CDR3 and/or
a VL
CDR3 of contained SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or
1881 -
1908 as disclosed in Table 1. Most preferably, antibodies of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six or more CDRs from
the same scFv,
as referred to in Table 1. The antibodies of the invention may be from any
animal origin,
including birds and mammals. Preferably, the antibodies are human, murine
(e.g., mouse
and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.
Most
preferably, the antibodies are human antibodies. As used herein, "human"
antibodies
include antibodies having the amino acid sequence of a human immunoglobulin
and
include antibodies isolated from human irnmunoglobulin libraries and xenomice
or other
organisms that have been genetically engineered to produce human antibodies.
For a
detailed discussion of a few of the technologies for producing human
antibodies and
human monoclonal antibodies and protocols for producing such antibodies, see,
e.g., PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European
Patent No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425;
5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; and
Lonberg and
Huszar, Int. Rev. Immunol. 13:65-93 (1995), which are incorporated by
reference herein
in their entirety. Human antibodies or "humanized" chimeric monoclonal
antibodies can
be produced using techniques described herein or otherwise known in the art.
For
example, methods for producing chimeric antibodies are known in the art. See,
for review
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the following references which are hereby incorporated in their entirety:
Morrison, Science
229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S.
Patent No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger
et al., WO
8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);
Neuberger et al., Nature 314:268 (1985). In addition, companies such as
Abgenix, Inc.
(Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide ,human
antibodies directed against a selected antigen using technology similar to
that described
above.
[0214] The antibodies of the present invention may be monovalent, bivalent,
trivalent
or multivalent. For example, monovalent scFvs can be multimerized either
chemically or
by association with another protein or substance. An scFv that is fused to a
hexahistidine
tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using
anti-Flag
antibodies (Stratagene, Inc.).
[0215] The antibodies of the present invention may be monospecific,
bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a BLyS polypeptide, or fragment thereof, or may be
specific for both
a BLyS polypeptide, or fragment thereof, and a heterologous epitope, such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-
69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819;
Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0216] The antibodies of the invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) may bind
immunospecifically to murine BLyS (e.g., a polypeptide having the amino acid
sequence
of human BLyS (SEQ ID NOS:3228 and/or 3229) or BLyS expressed on human
monocytes; murine BLyS (SEQ 117 NOS:3230 and/or 3231) or BLyS expressed on
murine
monocytes; rat BLyS (either the soluble forms as given in SEQ ID NOS:3232,
3233, 3234
and/or 3235 or in a membrane associated form, e.g., on the surface of rat
monocytes); or
monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NOS:3236 and/or
3237,
the soluble form of monkey BLyS, or BLyS expressed on monkey monocytes),
preferably
the antibodies of the invention bind immunospecifically to human BLyS.
Preferably, the
antibodies of the invention bind immunospecifically to human and monkey BLyS.
Also
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preferably, the antibodies of the invention bind immunospecifically to human
BLyS and
murine BLyS. More preferably, antibodies of the invention, bind
immunospecifically and
with higher affinity to human BLyS than to murine BLyS.
[0217] Antibodies of the present invention may also be described or specified
in terms
of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or
homolog of a polypeptide of the present invention are included. Antibodies
that bind
polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at
least 75%, at
least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity
(as calculated
using methods known in the art and described herein) to a polypeptide of the
present
invention are also included in the present invention. In a specific
embodiment, antibodies
of the present invention cross react with APRIL (SEQ ID N0:3239; GenBank
Accession
No. AF046888; J. Exp. Med. 188(6):1185-1190; PCT International Publication
W097/33902). In specific embodiments, antibodies of the present invention
cross-react
with murine, rat and/or rabbit homologs of human proteins and the
corresponding epitopes
thereof. Antibodies that do not bind polypeptides with less than 95%, less
than 90%, less
than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less
than 60%, less
than 55%, and less than 50% identity (as calculated using methods known in the
art and
described herein) to a polypeptide of the present invention are also included
in the present
invention. In a specific embodiment, the above-described cross-reactivity is
with respect
to any single specific antigenic or immunogenic polypeptide, or combinations)
of 2, 3, 4,
5, or more of the specific antigenic and/or immunogenic polypeptides disclosed
herein.
Further included in the present invention are antibodies which bind
polypeptides encoded
by polynucleotides which hybridize to a polynucleotide of the present
invention under
hybridization conditions (as described herein).
[0218] In a specific embodiment, antibodies of the present invention cross
react with
APRIL (SEQ ID N0:3239; GenBank Accession No. AF046888; J. Exp. Med.
188(6):1185-1190; PCT International Publication W097/33902). In specific
embodiments, antibodies that immunospecifically bind both BLyS and APRIL
comprise
all or a portion the BAB2001, BAB2080, BAB2015, BAB2019, BAB2087, BAB2016,
BAB2034 or BAB2065 scFVs (SEQ ID NOS:3240-3247). These scFvs were isolated by
panning a phage scFv library comprising VH and VL domains obtained from human
bone
marrow B cells (BM library). Phage from the BM phage library were first
selected for
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binding to soluble BLyS (amino acids 134-285 of SEQ ID N0:3228). A second
round of
selection for binding to the soluble form of APRIL (amino acids 105-250 of SEQ
ID
N0:3239) was then performed on the BLyS binding phage selected in round one. A
third
round of selection for binding to the soluble form of APRIL (amino acids 105-
250 of SEQ
ID N0:3239) was then performed on the phage selected in round two. A final
(fourth)
round of selection for binding to the soluble form of BLyS (amino acids 134-
285 of. SEQ
m N0:3228) was then performed on the phage selected in round three. Phage
clones that
bound BLyS in the fourth round of selection were eluted with either O.1M
triethylamine
(TEA) or with a TACI-Fc fusion protein (e.g., the extracellular domain of TACI
(amino
acids 31 to 159 of Genbank Accession No. AAC51790) fused to Fc). Eluted Phage
were
collected and sequenced (SEQ 117 NOS:3240-3247). Of 79 sequences, there were 8
unique sequences (SEQ ID NOS:3240-3247).
[0219] Isolated scFv clones (e.g., scFvs corresponding to SEQ ID NOS:3240-3247
or
other scFvs described in Table 1) or antibodies comprising at least a portion
of said scFV
clones may be screened for their ability to bind their ability to bind the
soluble form of
BLyS and the soluble form of APRIL by ELISA. Isolated scFv clones or
antibodies
comprising at least a portion of said scFv clones may also be screened for
their ability to
inhibit binding of a soluble form of BLyS or BLyS heterotrimer to TACI, BCMA
or
BAFF-R (Genbank Accession Nos. AAC51790, NP_00183, and NP 443177,
respectively). Isolated scFv clones or antibodies comprising at least a
portion of said
scFV clones may also be screened for their ability to inhibit BLyS or BLyS
heterotrimer
mediated biological activities (e.g. stimulation of B cell proliferation
and/or stimulation of
immunoglobulin production).
[0220] In specific embodiments, antibodies that immunospecifically bind both
BLyS
and APRIL comprise all or a portion (e.g., VHCDR, VLCDR, VH domain, VL domain)
of
the BAB2001, BAB2015, BAB2016, BAB2019, BAB2034, BAB2065, or BAB2080
scFVs (SEQ ID NOS:3240-3247).
[0221] In preferred embodiments, the antibodies of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), immunospecifically bind to BLyS and do not cross-react with any
other antigens.
In more preferred embodiments, the antibodies of the invention
immunopecifically bind to
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BLyS and do not cross-react with TRAIL, APRIL, Endokine-alpha, TNF-alpha, TNF-
beta,
Fas-L or LIGHT.
[0222] The present invention also provides for a nucleic acid molecule,
generally
isolated, encoding an antibody of the invention (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof). In one
embodiment, a
nucleic acid molecule of the invention encodes an antibody comprising, or
alternatively
consisting of, a VH domain having an amino acid sequence of any one of the VH
domains
referred to in Table 1. In another embodiment, a nucleic acid molecule of the
present
invention encodes an antibody comprising, or alternatively consisting of, a VH
CDR1
having an amino acid sequence of any one of the VH CDRls referred to in Table
1. In
another embodiment, a nucleic acid molecule of the present invention encodes
an antibody
comprising, or alternatively consisting of, a VH CDR2 having an amino acid
sequence of
any one of the VH CDR2s referred to in Table 1. In yet another embodiment, a
nucleic
acid molecule of the present invention encodes an antibody comprising, or
alternatively
consisting of, a VH CDR3 having an amino acid sequence of any one of the VH
CDR3s
referred to in Table 1. Nucleic acid molecules encoding antibodies that
immunospecifically bind BLyS and comprise, or alternatively consist of,
fragments or
variants of the VH domains and/or VH CDRs are also encompassed by the
invention:
[0223] In another embodiment, a nucleic acid molecule of the invention encodes
an
antibody (including molecules comprising, or alternatively consisting of,
antibody
fragments or variants thereof), comprising, or alternatively consisting of, a
VL domain
having an amino acid sequence of any one of the VL domains referred to in
Table 1. In
another embodiment, a nucleic acid molecule of the present invention encodes
an antibody
comprising, or alternatively consisting of, a VL CDR1 having amino acid
sequence of any
one of the VL CDRls referred to in Table 1. In another embodiment, a nucleic
acid
molecule of the present invention encodes an antibody comprising, or
alternatively
consisting of, a VL CDR2 having an amino acid sequence of any one of the VL
CDRZs
referred to in Table 1. In yet another embodiment, a nucleic acid molecule of
the present
invention encodes an antibody comprising, or alternatively consisting of, a VL
CDR3
having an amino acid sequence of any one of the VL CDR3s referred to in Table
1.
Nucleic acid encoding antibodies that immunospecifically bind BLyS and
comprise, or
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alternatively consist of, fragments or variants of the VL domains and/or
VLCDR(s) are
also encompassed by the invention.
[0224] In another embodiment, a nucleic acid molecule of the invention encodes
an
antibody (including molecules comprising, or alternatively consisting of,
antibody
fragments or variants thereof), comprising, or alternatively consisting of, a
VH domain
having an amino acid sequence of any one of the VH domains referred to in
Table 1 and a
VL domain having an amino acid sequence of any one of the VL domains referred
to in
Table 1. In another embodiment, a nucleic acid molecule of the invention
encodes an
antibody comprising, or alternatively consisting of, a VH CDR1, a VL CDRl, a
VH
CDR2, a VL CDR2, a VH CDR3, a VL CDR3, or any combination thereof having an
amino acid sequence referred to in Table 1. Nucleic acid encoding antibodies
that
immunospecifically bind BLyS and comprise, or alternatively consist of,
fragments or
variants of the VL andlor domains and/or VHCDR(s) and/or VLCDR(s) are also
encompassed by the invention.
[0225] The present invention also provides antibodies that comprise, or
alternatively
consist of, variants (including derivatives) of the VH domains, VH CDRs, VL
domains,
and VL CDRs described herein, which antibodies immunospecifically bind to
BLyS.
Standard techniques known to those of skill in the art can be used to
introduce mutations
in the nucleotide sequence encoding a molecule of the invention, including,
for example,
site-directed mutagenesis and PCR-mediated mutagenesis which result in amino
acid
substitutions. Preferably, the variants (including derivatives) encode less
than 50 amino
acid substitutions, less than 40 amino acid subsitutions, less than 30 amino
acid
substitutions, less than 25 amino acid substitutions, less than 20 amino acid
substitutions,
less than 15 amino acid substitutions, less than 10 amino acid substitutions,
less than 5
amino acid substitutions, less than 4 amino acid substitutions, less than 3
amino acid
substitutions, or less than 2 amino acid substitutions relative to the
reference VH domain,
VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDRl, VLCDR2, or VLCDR3. In
specific embodiments, the variants encode substitutions of VHCDR3. In a
preferred
embodiment, the variants have conservative amino acid substitutions at one or
more
predicted non-essential amino acid residues. A "conservative amino acid
substitution" is
one in which the amino acid residue is replaced with an amino acid residue
having a side
chain with a similar charge. Families of amino acid residues having side
chains with
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similar charges have been defined in the art. These families include amino
acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid,
glutarnic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains
e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine,
tryptophan, histidine). Alternatively, mutations can be introduced randomly
along all or
part of the coding sequence, such as by saturation mutagenesis, and the
resultant mutants
can be screened for biological activity to identify mutants that retain
activity (e.g., the
ability to bind BLyS). Following mutagenesis, the encoded protein may
routinely be
expressed and the functional and/or biological activity of the encoded
protein, (e.g., ability
to immunospecifically bind BLyS) can be determined using techniques described
herein or
by routinely modifying techniques known in the art.
[0226] The antibodies of the invention include derivatives (i.e., variants)
that are
modified, e.g., by the covalent attachment of any type of molecule to the
antibody such
that covalent attachment does not affect the ability of the antibody to
immunospecifically
bind to BLyS. For example, but not by way of limitation, derivatives of the
invention
include antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups,
proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous
chemical modifications may be carried out by known techniques, including, but
not
limited to, specific chemical cleavage, acetylation, formylation, metabolic
synthesis of
tunicamycin, etc. Additionally, the derivative may contain one or more non-
classical
amino acids.
[0227] In a specific embodiment, an antibody of the invention (including a
molecule
comprising, or alternatively consisting of, an antibody fragment or variant
thereof), that
immunospecifically binds BLyS, comprises, or alternatively consists of, an
amino acid
sequence encoded by a nucleotide sequence that hybridizes to a nucleotide
sequence that
is complementary to that encoding one of the VH or VL domains referred to in
Table 1
under stringent conditions, e.g., hybridization to filter-bound DNA in 6x
sodium
chloride/sodium citrate (SSC) at about 45° C followed by one or more
washes in
0.2xSSC/0.1% SDS at about 50-65° C, under highly stringent conditions,
e.g.,
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hybridization to filter-bound nucleic acid in 6xSSC at about 45° C
followed by one or
more washes in O.IxSSC/0.2% SDS at about 68° C, or under other
stringent hybridization
conditions which are known to those of skill in the art (see, for example,
Ausubel, F.M. et
al., eds. , 1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing
Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6
and 2.10.3).
In another embodiment, an antibody of the invention that immunospecifically
binds to
BLyS, comprises, or alternatively consists of, an amino acid sequence encoded
by a
nucleotide sequence that hybridizes to a nucleotide sequence that is
complementary to that
encoding one of the VH CDRs or VL CDRs referred to in Table 1 under stringent
conditions, e.g., hybridization under conditions as described above, or under
other
stringent hybridization conditions which are known to those of skill in the
art. In another
embodiment, an antibody of the invention that immunospecifically binds to
BLyS,
comprises, or alternatively consists of, an amino acid sequence encoded by a
nucleotide
sequence that hybridizes to a nucleotide sequence that is complementary to
that encoding
one of the VH CDR3s referred to in Table 1 under stringent conditions e.g.,
hybridization
under conditions as described above, or under other stringent hybridization
conditions
which are known to those of skill in the art. Nucleic acid molecules encoding
these
antibodies are also encompassed by the invention.
[0228] In another embodiment, an antibody (including a molecule comprising, or
alternatively consisting of, an antibody fragment or variant thereof), that
immunospecifically binds to BLyS comprises, or alternatively consists of, a
polypeptide
having an amino acid sequence that is at least 35%, at least 40%, at least
45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% identical, to any one
of the VH
domains referred to in Table 1. In another embodiment, an antibody of the
invention that
immunospecifically binds to BLyS comprises, or alternatively consists of, a
polypeptide
having an amino acid sequence that is at least 35%, at least 40%, at least
45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% identical, to any one
of the VH
CDRs referred to in Table 1. In another embodiment, an antibody of the
invention that
immunospecifically binds to BLyS comprises, or alternatively consists of, a
polypeptide
having an amino acid sequence that is at least 35%, at least 40%, at least
45%, at least
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50%, at least 55%, at least 60°70, at least 65%, at least 70%, at least
75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% identical to any one of
the VH
CDR3s referred to in Table 1. Nucleic acid molecules encoding these antibodies
are also
encompassed by the invention.
[0229] In another embodiment, an antibody of the invention (including a
molecule
comprising, or alternatively consisting of, an antibody fragment or variant
thereof), that
immunospecifically binds to BLyS comprises, or alternatively consists of, a
polypeptide
having an amino acid sequence that is at least 35%, at least 40%, at least
45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% identical, to any one
of the VL
domains referred to in Table 1. In another embodiment, an antibody of the
invention that
immunospecifically binds to BLyS comprises, or alternatively consists of, a
polypeptide
having an amino acid sequence that is at least 35%, at least 40%, at least
45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% identical, to any one
of the VL CDRs
referred to in Table 1. In another embodiment, an antibody of the invention
that
immunospecifically binds to BLyS comprises, or alternatively consists of, a
polypeptide
having an amino acid sequence that is at least 35%, at least 40°70, at
least 45%, at least
50%, at least 55%, at least 60010, at least 65%, at least 70%, at least 75%,
at least 80%, at
least 85%; at least 90%, at least 95%, or at least 99% identical, to any one
of the VL
CDR3s referred to in Table 1. Nucleic acid molecules encoding these antibodies
are also
encompassed by the invention.
[0230] Antibodies of the present invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) may also
be described
or specified in terms of their binding affinity for to BLyS polypeptides or
fragments or
variants of BLyS polypeptides (e.g., to the soluble form of BLyS and/or
membrane-bound
form of BLyS). In specific embodiments, antibodies of the invention bind BLyS
polypeptides, or fragments or variants thereof, with a dissociation constant
or KD of less
than or equal to 5 X 10-2 M, 10-2 M, 5 X 10-3 M,10-3 M, 5 X 10-4 M, 10-4 M, 5
X 10-5 M, or
10-5 M. More preferably, antibodies of 'the invention bind BLyS polypeptides
or
fragments or variants thereof with a dissociation constant or KD less than or
equal to 5 X
10-6 M, 10-6 M, 5 X 10-7 M, 10-7 M, 5 X 10-8 M, or 10-8 M. Even more
preferably,
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antibodies of the invention bind BLyS polypeptides or fragments or variants
thereof with a
dissociation constant or KD less than or equal to 5 X 10-9 M,10-9 M, 5 X 10-
1° M, 10-1° M,
X 10-11 M, 10-11 M, 5 X 10-12 M, 10-12 M, 5 X -13 M,10-is M, 5 X 10-14 M, 10-
14 M, 5 X
10-15 M, or 10-15 M. The invention encompasses antibodies that bind BLyS
polypeptides
with a dissociation constant or KD that is within any one of the ranges that
are between
each of the individual recited values.
[0231] In specific embodiments, antibodies of the invention bind BLyS
polypeptides
or fragments or variants thereof with an off rate (ko~) of less than or equal
to 5 X 10-2 sec
1, 10-Z sec 1, 5 X 10-3 sec 1 or 10-3 sec 1. More preferably, antibodies of
the invention bind
BLyS polypeptides or fragments or variants thereof with an off rate (kn~) less
than or
equal to 5 X 10-4 sec 1, 10-4 sec 1, 5 X 10-5 sec 1, or 10-5 sec-15 X 10-6 sec-
l, 10-6 sec 1, 5 X
10-7 sec 1 or 10-7 sec 1. The invention encompasses antibodies that bind BLyS
polypeptides
with an off rate (ko~) that is within any one of the ranges that are between
each of the
individual recited values.
[0232] In other embodiments, antibodies of the invention bind BLyS
polypeptides or
fragments or variants thereof with an on rate (l~n) of greater than or equal
to 103 M-1 sec 1,
5 X 103 M-1 sec 1, 104 M-1 sec 1 or 5 X 104 M-1 sec 1. More preferably,
antibodies of the
invention bind BLyS polypeptides or fragments or variants thereof with an on
rate (kon)
greater than or equal to 105 M-1 sec 1, 5 X 105 M-1 sec 1, 1061V>~1 sec l, or
5 X 106 M-1 sec 1
or 107 M-1 sec 1. The invention encompasses antibodies that bind BLyS
polypeptides with
on rate (kon) that is within any one of the ranges that are between each of
the individual
recited values.
[0233] The invention also encompasses antibodies (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) that
have one or
more of the same biological characteristics as one or more of the antibodies
described
herein. By "biological characteristics" is meant, the in vitro or in vivo
activities or
properties of the antibodies, such as, for example, the ability to bind to
BLyS (e.g., the
soluble form of BLyS, the membrane-bound form of BLyS, the soluble form and
membrane-bound form of BLyS), and/or an antigenic and/or epitope region of
BLyS), the
ability to substantially block BLySBLyS receptor (e.g., TACI - GenBank
accession
number AAC51790; BCMA - GenBank accession number ~ 001183; andlor BAFF-R -
GenBank accession number NP 443177) binding, or the ability to block BLyS
mediated
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biological activity (e.g., stimulation of B cell proliferation and
immunoglobulin
production). Optionally, the antibodies of the invention will bind to the same
epitope as at
least one of the antibodies specifically referred to herein. Such epitope
binding can be
routinely determined using assays known in the art.
[0234] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
neutralize BLyS or a fragment thereof, said antibodies comprising, or
alternatively
consisting of, a portion (i.e., a VH domain, VL domain, VH CDR1, VH CDR2, VH
CDR3, VL CDRl, VL CDR2, or VL CDR3) of an scFv referred to in Table l, more
preferably having an amino acid sequence contained in SEQ ID NOS:834 - 872,
1570
1595, or 1886 - 1908, and even more preferably having an amino acid sequence
contained
in SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, or 1881-1885 as disclosed in
Table 1, or
a fragment or variant thereof. By an antibody that "neutralizes BLyS or a
fragment
thereof ' is meant an antibody that diminishes or abolishes the ability of
BLyS to bind to
its receptor (e.g., TACI - GenBank accession number AAC51790; BCMA - GenBank
accession number NP_001183; andl'or -BAFF-R - GenBank accession number NP
443177)
to stimulate B cell proliferation, to stimulate immunoglobulin secretion by B
cells, and/or
to stimulate the BLyS receptor signalling cascade. In one embodiment, an
antibody that
neutralizes BLyS or a fragment thereof, comprises, or alternatively consists
of, a
polypeptide having the amino acid sequence of a VH domain contained in SEQ ID
NOS:l
- 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table
1, or a
fragment or variant thereof. In another embodiment, an antibody that
neutralizes BLyS or
a fragment thereof, comprises, or alternatively consists of, a polypeptide
having the amino
acid sequence of a VL domain contained in SEQ ID NOS:l - 46, 321 - 329, 834 -
872,
1563 - 1595, or 1881 - 1908 as disclosed in Table 1, or a fragment or variant
thereof. In
another embodiment, an antibody that neutralizes BLyS or a fragment thereof,
comprises,
or alternatively consists of, a polypeptide having the amino acid sequence of
a VH CDR
domain in SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908
as
disclosed in Table 1, or a fragment or variant thereof. In a preferred
embodiment, an
antibody that neutralizes BLyS or a fragment thereof, comprises, or
alternatively consists
of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ
ID
NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881-1908 as disclosed in
Table 1, or
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a fragment or variant thereof. In another embodiment, an antibody that
neutralizes BLyS
or a fragment thereof, comprises, or alternatively consists of, a polypeptide
having the
amino acid sequence of a VL CDR domain contained in SEQ ID NOS:1 - 46, 321 -
329,
834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table l, or,a fragment
or variant
thereof. In another preferred embodiment, an antibody that neutralizes BLyS or
a
fragment thereof, comprises, or alternatively consists of, a polypeptide
having the amino
acid sequence of a VL CDR3 contained in SEQ ID NOS:1 - 46, 321- 329, 834 -
872, 1563
- 1595, or 1881 - 1908 as disclosed in Table 1, or a fragment or variant
thereof. Nucleic
acid molecules encoding these antibodies are also encompassed by the
invention.
[0235] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
inhibit (i.e., diminish or abolish) BLyS-mediated B cell proliferation as
determined by any
method known in the art such as, for example, the assays described in Examples
21 and
22, infra, said antibodies comprising, or alternatively consisting of, a
portion (e.g., a VH
domain, VL domain, VH CDRl, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL
CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834 - 872, 1570 -
1595,
1886 - 1908, and even more preferably having an amino acid sequence SEQ ID
NOS:l -
46, 321 - 329, 1563 - 1569, 1881 - 1885 as disclosed in Table 1 or a fragment
or variant
thereof. In one embodiment, an antibody that inhibits BLyS-mediated B cell
proliferation, comprises, or alternatively consists of, a polypeptide having
the amino acid
sequence of a VH domain contained in SEQ ID NOS:1 - 46, 321 - 329, 834 - 872,
1563 -
1595, or 1881 - 1908, as disclosed in Table 1, or a fragment or variant
thereof. In another
embodiment, an antibody that inhibits BLyS-mediated B cell proliferation,
comprises, or
alternatively consists of, a polypeptide having the amino acid sequence of a
VL domain
contained in SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 -
1908 as
disclosed in Table l, or a fragment or variant thereof. In a preferred
embodiment, an
antibody that inhibits BLyS-mediated B cell proliferation, comprises, or
alternatively
consists of, a polypeptide having the amino acid sequence of a VH CDR3
contained in
SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as
disclosed in
Table 1, or a fragment or variant thereof. In another preferred embodiment, an
antibody
that inhibits BLyS-mediated B cell proliferation, comprises, or alternatively
consists of, a
polypeptide having the amino acid sequence of a VL CDR3 contained SEQ DJ NOS:1
-
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46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1,
or a
fragment or variant thereof. Nucleic acid molecules encoding these antibodies
are also
encompassed by the invention.
[0236] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
inhibit (i.e., diminish or abolish) BLyS-mediated stimulation of B cell
survival as
determined by any method known in the art such as, for example, the assays
described in
Examples 21 and 22, infra, said antibodies comprising, or alternatively
consisting of, a
portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1,
VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834 -
872, 1570 - 1595, 1886 - 1908, and even more preferably having an amino acid
sequence
SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, 1881 - 1885 as disclosed in Table 1
or a
fragment or variant thereof. In one embodiment, an antibody that inhibits BLyS-
mediated
stimulation of B cell survival, comprises, or alternatively consists of, a
polypeptide
having the amino acid sequence of a VH domain contained in SEQ ID NOS:1 - 46,
321 -
329, 834 - 872, 1563 - 1595, or 1881 - 1908, as disclosed in Table 1, or a
fragment or
variant thereof. In another embodiment, an antibody that inhibits BLyS-
mediated
stimulation of B cell survival, comprises, or alternatively consists of, a
polypeptide having
the amino acid sequence of a VL domain contained in SEQ ID NOS:1 - 46, 321 -
329, 834
- 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1, or a fragment or
variant
thereof. In a preferred embodiment, an antibody that inhibits BLyS-mediated
stimulation
of B cell survival, comprises, or alternatively consists of, a polypeptide
having the amino
acid sequence of a VH CDR3 contained in SEQ ID NOS:1 - 46, 321 - 329, 834 -
872,
1563 - 1595, or 1881 - 1908 as disclosed in Table 1, or a fragment or variant
thereof. In
another preferred embodiment, an antibody that inhibits BLyS-mediated
stimulation of B
cell survival, comprises, or alternatively consists of, a polypeptide having
the amino acid
sequence of a VL CDR3 contained SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 -
1595, or 1881 - 1908 as disclosed in Table 1, or a fragment or variant
thereof. Nucleic
acid molecules encoding these antibodies are also encompassed by the
invention.
[0237] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
inhibit (i.e., diminish or abolish) BLyS-mediated stimulation of B cell
differentiation as
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determined by any method known in the art such as, for example, the assays
described in
Examples 21 and 22, infra, said antibodies comprising, or alternatively
consisting of, a
portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1,
VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834 -
872, 1570 - 1595, 1886 - 1908, and even more preferably having an amino acid
sequence -,
SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, 1881 - 1885 as disclosed in Table 1
or a
fragment or variant thereof. In one embodiment, an antibody that inhibits BLyS-
mediated
stimulation of B cell differentiation, comprises, or alternatively consists
of, a polypeptide
having the amino acid sequence of a VH domain contained in SEQ ID NOS:1 - 46,
321 -
329, 834 - 872, 1563 - 1595, or 1881 - 1908, as disclosed in Table 1, or a
fragment or
variant thereof. In another embodiment, an antibody that inhibits BLyS-
mediated
stimulation of B cell differentiation, comprises, or alternatively consists
of, a polypeptide
having the amino acid sequence of a VL domain contained in SEQ ID NOS:l - 46,
321 -
329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table l, or a
fragment or
variant thereof. In a preferred embodiment, an antibody that inhibits BLyS-
mediated
stimulation of B cell differentiation, comprises, or alternatively consists
of, a polypeptide
having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1 - 46,
321 -
329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table l, or a
fragment or
variant thereof. In another preferred embodiment, an antibody that inhibits
BLyS-
mediated stimulation of B cell differentiation, comprises, or alternatively
consists of, a
polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS:1
-
46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1,
or a
fragment or variant thereof. Nucleic acid molecules encoding these antibodies
are also
encompassed by the invention.
[0238] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
inhibit (i.e., diminish or abolish) BLyS-mediated stimulation of
immunoglobulin
production by B cells as determined by any method known in the art such as,
for example,
the assays described in Examples 21 and 22, infra, said antibodies comprising,
or
alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDRl,
VH
CDR2, VH CDR3, VL CDRl, VL CDR2, or VL CDR3) of an scFv having an amino acid
sequence SEQ ID NOS:834 - 872, 1570 - 1595, 1886 - 1908, and even more
preferably
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having an amino acid sequence SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, 1881 -
1885
as disclosed in Table 1 or a fragment or variant thereof. In one embodiment,
an antibody
that inhibits BLyS-mediated stimulation of immunoglobulin production by B
cells,
comprises, or alternatively consists of, a polypeptide having the amino acid
sequence of a
VH domain contained in SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595,
or 1881
-1908, as disclosed in Table 1, or a fragment or variant thereof. In another
embodiment,
an antibody that inhibits BLyS-mediated stimulation of immunoglobulin
production by B
cells, comprises, or alternatively consists of, a polypeptide having the amino
acid
sequence of a VL domain contained in SEQ ID NOS:l - 46, 321 - 329, 834 - 872,
1563 -
1595, or 1881 - 1908 as disclosed in Table l, or a fragment or variant
thereof. In a
preferred embodiment, an antibody that inhibits BLyS-mediated stimulation of
immunoglobulin production by B cells, comprises, or alternatively consists of,
a
polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID
NOS:1 -
46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1,
or a
fragment or variant thereof. In another preferred embodiment, an antibody that
inhibits
BLyS-mediated stimulation of immunoglobulin production by B cells, comprises,
or
alternatively consists of, a polypeptide having the amino acid sequence of a
VL CDR3
contained SEQ ID NOS:l - 46, 321 ~- 329, 834 - 872, 1563 - 1595, or 1881 -
1908 as
disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules
encoding
these antibodies are also encompassed by the invention.
[0239] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
enhance the activity of BLyS or a fragment thereof, said antibodies
comprising, or
alternatively consisting of, a portion (i.e., a VH domain, VL domain, VH CDR1,
VH
CDR2, VH CDR3, VL CDRl, VL CDR2, or VL CDR3) of an scFv having an amino acid
sequence SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 - 1908, and preferably
having an
amino acid sequence of SEQ ID NOS:1 - 46, 321 - 329, 1563 - 1569, or 1881 -
1885, as
disclosed in Table 1, or a fragment or variant thereof. By an antibody that
"enhances the
activity of BLyS or a fragment thereof ' is meant an antibody increases the
ability of BLyS
to bind to its receptor (e.g., TACI - GenBank accession number AAC51790; BCMA -
GenBank accession number NP-001183; and/or BAFF-R - GenBank accession number
TTp 443177), to stimulate B cell proliferation, to stimulate immunoglobulin
secretion by B
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cells, and/or to stimulate the BLyS receptor signalling cascade. In one
embodiment, an
antibody that enhances the activity of BLyS or a fragment thereof, comprises,
or
alternatively consists of, a polypeptide having the amino acid sequence of a
VH domain
contained in SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 -
1908 as
disclosed in Table l, or a fragment or variant thereof. In another embodiment,
an antibody
that enhances the activity of BLyS or a fragment thereof, comprises, or
alternatively
consists of, a polypeptide having the amino acid sequence of a VL domain
contained in
SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as
disclosed in
Table 1, or a fragment or variant thereof. In another embodiment, an antibody
that
enhances the activity of BLyS or a fragment thereof, comprises, or
alternatively consists
of, a polypeptide having the amino acid sequence of a VH CDR domain contained
in SEQ
ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881-1908 as disclosed in
Table 1,
or a fragment or variant thereof. In a preferred embodiment, an antibody that
enhances the
activity of BLyS or a fragment thereof, comprises, or alternatively consists
of, a
polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID
NOS:1 -
46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1,
or a
fragment or variant thereof. In another embodiment, an antibody that enhances
BLyS or a
fragment thereof, comprises, or alternatively consists of, a polypeptide
having the amino
acid sequence of a VL CDR domain contained in SEQ m NOS:1 - 46, 321 - 329, 834
-
872, 1563 - 1595, or 1881- 1908 as disclosed in Table l, or a fragment or
variant thereof.
In another preferred embodiment, an antibody that enhances the activity of
BLyS or a
fragment thereof, comprises, or alternatively consists of, a polypeptide
having the amino
acid sequence of a VL CDR3 contained in SEQ ID NOS:1- 46, 321 - 329, 834 -
872, 1563
- 1595, or 1881 - 1908 as disclosed in Table 1, or a fragment or variant
thereof. Nucleic
acid molecules encoding these antibodies are also encompassed by the
invention.
[0240] The present invention also provides for antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
stimulate BLyS-mediated B cell proliferation as determined by any method known
in the
art, such as, for example, the assays described in Examples 21 and 22, infra,
said
antibodies comprising, or alternatively consisting of, a portion (e.g., a VH
domain, VL
domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an
scFv having an amino acid sequence of SEQ ID NOS:834 - 872, 1570 - 1595, or
1886 -
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1908, and even more preferably having an amino acid sequence of SEQ 117 NOS:1 -
46,
321 - 329, 1563 - 1569, or 1881 - 1885 as disclosed in Table 1 or a fragment
or variant
thereof. In one embodiment, an antibody that stimulates BLyS-mediated B cell
proliferation, comprises, or alternatively consists of, a polypeptide having
the amino acid
sequence of a VH domain contained in SEQ ID NOS:1 - 46, 321 - 329, 834 - 872,
1563 -
1595, or 1881 - 1908 as disclosed in Table l, or a fragment or variant
thereof. In another
embodiment, an antibody that stimulates BLyS-mediated B cell proliferation,
comprises,
or alternatively consists of, a polypeptide having the amino acid sequence of
a VL domain
contained in SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 -
1908 as
disclosed in Table 1, or a fragment or variant thereof. In a preferred
embodiment, an
antibody that stimulates BLyS-mediated B cell proliferation, comprises, or
alternatively
consists of, a polypeptide having the amino acid sequence of a VH CDR3
contained in
SEQ ID NOS:1 - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as
disclosed in
Table 1, or a fragment or variant thereof. In another preferred embodiment, an
antibody
that stimulates BLyS-mediated B cell proliferation, comprises, or
alternatively consists of,
a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID
NOS:1
- 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table
1, or a
fragment or variant thereof. Nucleic acid molecules encoding these antibodies
are also
encompassed by the invention.
[0241] The present invention also provides for fusion proteins comprising, or
alternatively consisting of, an antibody (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof), that
immunospecifically binds to
BLyS, and a heterologous polypeptide. Preferably, the heterologous polypeptide
to which
the antibody is fused to is useful for B-cell function or is useful to target
the antibody to B-
cells. In an alternative preferred embodiment, the heterologous polypeptide to
which the
antibody is fused to is useful for monocyte cell function or is useful to
target the antibody
to a monocyte. In another embodiment, the heterologous polypeptide to which
the
antibody is fused is albumin (including but not limited to recombinant human
serum
albumin or fragments or variants thereof (see, e.g., U.S. Patent No.
5,876,969, issued
March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June
16, 1998,
herein incorporated by reference in their entirety)). In a preferred
embodiment, antibodies
of the present invention (including fragments or variants thereof) are fused
with the
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mature form of human serum albumin (i.e., amino acids 1 - 585 of human serum
albumin
as shown in Figures 1 and 2 of EP Patent 0 322 094) which is herein
incorporated by
reference in its entirety. In another preferred embodiment, antibodies of the
present
invention (including fragments or variants thereof) are fused with polypeptide
fragments
comprising, or alternatively consisting of, amino acid residues 1-x of human
serum
albumin, where x is an integer from 1 to 585 and the albumin fragment has
human serum
albumin activity. In another preferred embodiment, antibodies of the present
invention
(including fragments or variants thereof) are fused with polypeptide fragments
comprising, or alternatively consisting of, amino acid residues 1-z of human
serum
albumin, where z is an integer from 369 to 419, as described in LT.S. Patent
5,766,883
herein incorporated by reference in its entirety. Antibodies of the present
invention
(including fragments or variants thereof) may be fused to either the N- or C-
terminal end
of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human
serum
albumin polypeptide).
[0242] In one embodiment, a fusion protein of the invention comprises, or
alternatively consists of, a polypeptide having the amino acid sequence of any
one or more
of the VH domains referred to in Table 1 or the amino acid sequence of any one
or more
of the VL domains referred to in Table 1 or fragments or variants thereof, and
a
heterologous polypeptide sequence. In another embodiment, a fusion protein of
the
present invention comprises, or alternatively consists of, a polypeptide
having the amino
acid sequence of any one, two, three, or more of the VH CDRs referred to in
Table 1, or
the amino acid sequence of any one, two, three, or more of the VL CDRs
referred to in
Table 1, or fragments or variants thereof, and a heterologous polypeptide
sequence. In a
preferred embodiment, the fusion protein comprises, or alternatively consists
of, a
polypeptide having the amino acid sequence of, a VH CDR3 referred to in Table
1, or
fragment or variant thereof, and a heterologous polypeptide sequence, which
fusion
protein immunospecifically binds to BLyS. In another embodiment, a fusion
protein
comprises, or alternatively consists of a polypeptide having the amino acid
sequence of at
least one VH domain referred to in Table 1 and the amino acid sequence of at
least one VL
domain referred to in Table 1 or fragments or variants thereof, and a
heterologous
polypeptide sequence. Preferably, the VH and VL domains of the fusion protein
correspond to the same scFv referred to in Table 1. In yet another embodiment,
a fusion
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protein of the invention comprises, or alternatively consists of a polypeptide
having the
amino acid sequence of any one, two, three or more of the VH CDRs referred to
in Table 1
and the amino acid sequence of any one, two, three or more of the VL CDRs
referred to in
Table l, or fragments or variants thereof, and a heterologous polypeptide
sequence.
Preferably, two, three, four, five, six, or more of the VHCDR(s) or VLCDR(s)
correspond
to the same scFv referred to in Table 1. Nucleic acid molecules encoding these
fusion
proteins are also encompassed by the invention.
[0243] The present invention also provides: antibodies (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), that
immunospecifically bind to the soluble form of BLyS; antibodies that
immunospecifically
bind to the membrane-bound form of BLyS; and antibodies that
immunospecifically bind
to both the soluble form and membrane-bound form of BLyS.
[0244] In one embodiment of the present invention, antibodies (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to the soluble form of BLyS, comprise, or
alternatively consist
of, a polypeptide having the amino acid sequence of any one or more of the VH
domains
contained in SEQ ID NOS:1563 - 1880 as disclosed in Table 1 and/or the amino
acid
sequence of any one or more of the VL domains contained in SEQ ID NOS: 1563 -
1880
as disclosed in Table 1, or fragments) or variants) (including derivative)
thereof.
Preferably, the VH and VL domains of the antibody correspond to the same scFv
as
disclosed in Table 1. In another embodiment, antibodies that
immunospecifically bind to
the soluble form of BLyS are provided- that comprise, or alternatively consist
of, a
polypeptide having the amino acid sequence of any one, two, three, or more of
the VH
CDRs contained SEQ ID NOS: 1563 - 1880 as disclosed in Table 1 and/or the
amino acid
sequence of any one, two, three, or more of the VL CDRs contained in SEQ ID
NOS:
1563 - 1880 as disclosed in Table l, or fragments) or variants) thereof.
Preferably, two,
three, four, five, six or more of the VH and VL CDRs of the antibody
correspond to the
same scFv as disclosed in Table 1. In a preferred embodiment, antibodies that
immunospecifically bind to the soluble form of BLyS are provided that
comprise, or
alternatively consist of, a polypeptide having the amino acid sequence of any
one or more
of the VH CDR3s contained in SEQ ID NOS: 1563 - 1880 as disclosed in Table 1
and/or
the amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID
NOS:
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1563 - 1880 as disclosed in Table 1, or fragments) or variants) thereof.
Preferably, the
VHCDR3 and VLCDR3 of the antibody correspond to the same scFv, as disclosed in
Table 1. Nucleic acid molecules encoding these antibodies are also encompassed
by the
invention.
[0245] In another embodiment of the present invention, antibodies (including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof) that immunospecifically bind to the membrane-bound form of BLyS are
provided
that comprise, or alternatively consist of, a polypeptide having the amino
acid sequence of
any one or more of the VH domains contained in SEQ ID NOS: 1881 - 2128 as
disclosed
in Table 1 and/or the amino acid sequence of any one or more of the VL domains
contained in SEQ ID NOS: 1881 - 2128 as disclosed in Table 1, or a fragment or
variant
thereof. Preferably, the VH and VL domains of the antibody correspond to the
same scFv
as disclosed in Table 1. In another embodiment, antibodies that
immunospecifically bind
to the membrane-bound form of BLyS are provided that comprise, or
alternatively consist
of, a polypeptide having the amino acid sequence of any one, two, three, or
more of the
VH CDRs contained in SEQ ID NOS: 1881 - 2128 as disclosed in Table 1 and/or
the
amino acid sequence of any one, two, three, or more of the VL CDRs contained
in SEQ ID
NOS: 1881 - 2128 as disclosed in Table l, or fragments) or variants) thereof.
Preferably,
two, three, four, five, six or more of the VH and VL CDRs of the antibody
correspond to
the same scFv as disclosed in Table 1. In a preferred embodiment, antibodies
that
immunospecifically bind to the membrane-bound form of BLyS are provided that
comprise, or alternatively consist of, a polypeptide having the amino acid
sequence of any
one or more of the VH CDR3s contained in SEQ ID NOS: 1881 - 2128 as disclosed
in
Table 1 and/or the amino acid sequence of any one or more of the VL CDR3s
contained in
SEQ ID NOS: 1881 - 2128 as disclosed in Table 1, or fragments) or variants)
thereof.
Preferably, the VHCDR3 and VLCDR3 of the antibody correspond to the same scFv,
as
disclosed in Table 1. Nucleic acid molecules encoding these antibodies are
also
encompassed by the invention.
[0246] In another embodiment of the present invention, antibodies (including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof) that immunospecifically bind to the soluble form and membrane-bound
form of
BLyS, are provided that comprise, or alternatively consist of, a polypeptide
having the
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amino acid sequence of any one or more of the VH domains contained in SEQ ID
NOS: 1
- 1562 as disclosed in Table 1 and/or the amino acid sequence of any one or
more of the
VL domains contained in SEQ ID NOS: 1 - 1562 as disclosed in Table 1, or a
fragment or
variant thereof. Preferably, the VH and VL domains of the antibody correspond
to the
same scFv as disclosed in Table 1. In another embodiment, antibodies that
immunospecifically bind to the soluble form and membrane-bound form of BLyS
are
provided that comprise, or alternatively consist of, a polypeptide having the
amino acid
sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID
NOS: 1 -
1562 as disclosed in Table 1 and/or the amino acid sequence of any one, two,
three, or
more of the VL CDRs contained in SEQ ID NOS: 1 - 1562 as disclosed in Table 1,
or
fragments) or variants) thereof. Preferably, two, three, four, five, six or
more of the VH
and VL CDRs of the antibody correspond to the same scFv as disclosed in Table
1. In a
preferred embodiment, antibodies that immunospecifically bind to the soluble
form and
membrane-bound form of BLyS are provided that comprise, or alternatively
consist of, a
polypeptide having the amino acid sequence of any one or more of the VH CDR3s
contained in SEQ ID NOS: 1 - 1562, disclosed in Table 1 and/or the amino acid
sequence
of any one or more of the VL CDR3s contained in SEQ ID NOS: 1 - 1562,
disclosed in
Table l, or fragments) or variants) thereof. Preferably, the VHCDR3 and VLCDR3
of
the antibody correspond to the same scFv, as disclosed in Table 1.
[0247] The present invention also provides for mixtures of antibodies
(including scFvs
and other molecules comprising, or alternatively consisting of, antibody
fragments or
variants thereof) that immunospecifically bind to BLyS, wherein the mixture
has at least
one, two, three, four, five or more different antibodies of the invention. In
particular, the
invention provides for mixtures of different antibodies that
immunospecifically bind to the
soluble form of BLyS, the membrane-bound form of BLyS, and/or both the
membrane-
bound form and soluble form of BLyS. In specific embodiments, the invention
provides
mixtures of at least 2, preferably at least 4, at least 6, at least ~, at
least 10, at least 12, at
least 15, at least 20, or at least 25 different antibodies that
immunospecifically bind to
BLyS, wherein at least 1, at least 2, at least 4, at least 6, or at least 10,
antibodies of the
mixture is an antibody of the invention. In a specific embodiment, each
antibody of the
mixture is an antibody of the invention.
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[0248] The present invention also provides for panels of antibodies (including
scFvs
and other molecules comprising, or alternatively consisting of, antibody
fragments or
variants thereof) that immunospecifically bind to BLyS, wherein the panel has
at least one,
two, three, four, five or more different antibodies of the invention. In
particular, the
invention provides for panels of different antibodies that immunospecifically
bind to the
soluble form of BLyS, the membrane-bound form of BLyS, and/or both the
membrane-
bound form and soluble form of BLyS. In specific embodiments, the invention
provides
for panels of antibodies that have different affinities for BLyS, different
specificities for
BLyS, or different dissociation rates. The invention provides panels of at
least 10,
preferably at least 25, at least 50, at least 75, at least 100, at least 125,
at least 150, at least
175, at least 200, at least 250, at least 300, at least 350, at least 400, at
least 450, at least
500, at least 550, at least 600, at least 650, at least 700, at least 750, at
least 800, at least
850, at least 900, at least 950, or at least 1000, antibodies. Panels of
antibodies can be
used, for example, in 96 well plates for assays such as ELISAs.
[0249] The present invention further provides for compositions comprising, one
or
more antibodies (including scFvs and other molecules comprising, or
alternatively
consisting of antibody fragments or variants of the invention). In one
embodiment, a
composition of the present invention comprises, one, two, three, four, five,
or more
antibodies that comprise or alternatively consist of, a polypeptide having an
amino acid
sequence of any one or more of the VH domains contained in SEQ ID NOS:1563 -
1880
as disclosed in Table 1, or a variant thereof. In another embodiment, a
composition of the
present invention comprises, one, two, three, four, five, or more antibodies
that comprise,
or alternatively consist of, a polypeptide having an amino acid sequence of
any one or
more of the VH CDRls contained in SEQ ID NOS:1563 - 1880 as disclosed in Table
1, or
a variant thereof. In another embodiment, a composition of the present
invention
comprises, one, two, three, four, five or more antibodies that comprise, or
alternatively
consist of, a polypeptide having an amino acid sequence of any one or more of
the VH
CDR2s contained in SEQ T17 NOS:1563 - 1880 as disclosed in Table 1, or a
variant
thereof. In a preferred embodiment, a composition of the present invention
comprises,
one, two, three, four, five, or more antibodies that comprise, or
alternatively consist of, a
polypeptide having an amino acid sequence of any one or more of the VH CDR3s
contained in SEQ ID NOS:1563 - 1880, as disclosed in Table 1 or a variant
thereof.
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[0250] The present invention further provides for compositions comprising, one
or
more antibodies (including scFvs and other molecules comprising, or
alternatively
consisting of antibody fragments or variants of the invention). In one
embodiment, a
composition of the present invention comprises, one, two, three, four, five,
or more
antibodies that comprise or alternatively consist of, a polypeptide having an
amino acid
sequence of any one or more of the VH domains contained in SEQ ID NOS:1881 -
2128
as disclosed in Table 1, or a variant thereof. In another embodiment, a
composition of the
present invention comprises, one, two, three, four, five, or more antibodies
that comprise,
or alternatively consist of, a polypeptide having an amino acid sequence of
any one or
more of the VH CDRls contained in SEQ 117 NOS:1881 - 2128 as disclosed in
Table 1, or
a variant thereof. In another embodiment, a composition of the present
invention
comprises, one, two, three, four, five or more antibodies that comprise, or
alternatively
consist of, a polypeptide having an amino acid, sequence of any one or more of
the VH
CDR2s contained in SEQ ID NOS:1881 - 2128 as disclosed in Table l, or a
variant
thereof. In a preferred embodiment, a composition of the present invention
comprises,
one, two, three, four, five, or more antibodies that comprise, or
alternatively consist of, a
polypeptide having an amino acid sequence of any one or more of the VH CDR3s
contained in SEQ >D NOS:1881 - 2128 as disclosed in Table 1 or a variant
thereof.
[0251] The present invention further provides for compositions comprising, one
or
more antibodies (including scFvs, or molecules comprising, or alternatively
consisting of
antibody fragments or variants of the invention). In one embodiment, a
composition of the
present invention comprises, one, two, three, four, five, or more antibodies
that comprise
or alternatively consist of, a polypeptide having an amino acid sequence of
any one or
more of the VH domains contained in SEQ ID NOS:1 - 1562 as disclosed in Table
1, or a
variant thereof. In another embodiment, a composition of the present invention
comprises, one, two, three, four, five, or more antibodies that comprise, or
alternatively
consist of, a polypeptide having an amino acid sequence of any one or more of
the VH
CDRls contained in SEQ ID NOS:1 - 1562 as disclosed in Table 1, or a variant
thereof.
In another embodiment, a composition of the present invention comprises, one,
two, three,
four, five or more antibodies that comprise, or alternatively consist of, a
polypeptide
having an amino acid sequence of any one or more of the VH CDR2s contained in
SEQ
ID NOS:1 - 1562 as disclosed in Table l, or a variant thereof. In a preferred
embodiment,
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a composition of the present invention comprises, one, two, three, four, five,
or more
antibodies that comprise, or alternatively consist of, a polypeptide having an
amino acid
sequence of any one or more of the VH CDR3s contained in SEQ ID NOS:1 - 1562
as
disclosed in Table 1 or a variant thereof.
[0252] Other embodiments of the present invention providing for compositions
comprising, one or more antibodies (including scFvs and other molecules
comprising, or
alternatively consisting of antibody fragments or variants of the invention)
are listed
below. In another embodiment, a composition of the present invention
comprises, one,
two, three, four, five, or more antibodies that comprise, or alternative
consist of, a
polypeptide having an amino acid sequence of any one or more of the VL domains
contained in SEQ ID NOS:1563 - 1880 as disclosed in Table 1, or a variant
thereof. In
another embodiment, a composition of the present invention comprises, one,
two, three,
four, five, or more antibodies that comprise, or alternatively consist of, a
polypeptide
having an amino acid sequence of any one or more of the VL CDRls contained in
SEQ ID
NOS:1563 - 1880 as disclosed in Table 1, or a variant thereof. In another
embodiment, a
composition of the present invention comprises, one, two, three, four, five,
or more
antibodies that comprise, or alternatively consist of, a polypeptide having an
amino acid
sequence of any one or more of the VL CDR2s contained SEQ ID NOS:1563 - 1880
as
disclosed in Table 1, or a variant thereof. In a preferred embodiment, a
composition of the
present invention comprises, one, two, three, four, five, or more antibodies
that comprise,
or alternatively consist of, a polypeptide having an amino acid sequence of
any one or
more of the VL CDR3s contained in SEQ ID NOS:1563 - 1880 as disclosed in Table
1, or
a variant thereof.
[0253] Other embodiments of the present invention providing for compositions
comprising, one or more antibodies (including scFvs and other molecules
comprising, or
alternatively consisting of antibody fragments or variants of the invention)
are listed
below. In another embodiment, a composition of the present invention
comprises, one,
two, three, four, five, or more antibodies that comprise, or alternatively
consist of, a
polypeptide having an amino acid sequence of any one or more of the VL domains
contained in SEQ ID NOS:1881 - 2128 as disclosed in Table 1, or a variant
thereof. In
another embodiment, a composition of the present invention comprises, one,
two, three,
four, five, or more antibodies that comprise, or alternatively consist of, a
polypeptide
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having an amino acid sequence of any one or more of the VL CDRls contained in
SEQ ID
NOS:1881 - 2128 as disclosed in Table 1, or a variant thereof. In another
embodiment, a
composition of the present invention comprises, one, two, three, four, five,
or more
antibodies that comprise, or alternatively consist of, a polypeptide having an
amino acid
sequence of any one or more of the VL CDR2s SEQ ID NOS:1881 - 2128 as
disclosed in
Table l, or a variant thereof. In a preferred embodiment, a composition of the
present
invention comprises, one, two, three, four, five, or more antibodies that
comprise, or
alternatively consist of, a polypeptide having an amino acid sequence of any
one or more
of the VL CDR3s contained in SEQ ID NOS:1881 - 2128 as disclosed in Table 1,
or a
variant thereof.
[0254] Other embodiments of the present invention providing for compositions
comprising, one or more antibodies (including scFvs and other molecules
comprising, or
alternatively consisting of antibody fragments or variants of the invention)
are listed
below. In another embodiment, a composition of the present invention
comprises, one,
two, three, four, five, or more antibodies that comprise, or alternatively
consist of, a
polypeptide having an amino acid sequence of any one or more of the VL domains
contained in SEQ ID NOS:1 - 1562 as disclosed in Table 1, or a variant
thereof. In
another embodiment, a composition of the present invention comprises, one,
two, three,
four, five, or more antibodies that comprise, or alternatively consist of, a
polypeptide
having an amino acid sequence of any one or more of the VL CDRls contained in
SEQ ID
NOS:1 - 1562 as disclosed in Table l, or a variant thereof. In another
embodiment, a
composition of the present invention comprises, one, two, three, four, five,
or more
antibodies that comprise, or alternatively consist of, a polypeptide having an
amino acid
sequence of any one or more of the VL CDR2s SEQ ID NOS:1 - 1562 as disclosed
in
Table 1, or a variant thereof. In a preferred embodiment, a composition of the
present
invention comprises, one, two, three, four, five, or more antibodies that
comprise, or
alternatively consist of, a polypeptide having an amino acid sequence of any
one or more
of the VL CDR3s contained in SEQ ID NOS:1 - 1562 as disclosed in Table 1, or a
variant
thereof.
[0255] In a preferred embodiment, a composition of the present invention
comprises,
one, two, three, four, five, or more antibodies that comprise, or
alternatively consist of, a
polypeptide having an amino acid sequence of any one or more of the VH domains
in
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disclosed in Table 1, or a variant thereof, and an amino acid sequence of any
one or more
of the VL domains disclosed in Table 1, or a variant thereof wherein the VH
and VL
domains are from scFvs with the same specificity (i.e., from scFvs that bind
soluble BLyS
(SEQ ID NOS:1563 - 1880), from scFvs that bind membrane-bound BLyS (SEQ ID
1881
- 2128), or from scFvs that bind both soluble and membrane-bound BLyS (SEQ ID
NOS:1
- 1562). In a preferred embodiment the invention provides antibodies wherein
the VH
CDRX (where X=1,2, or 3) and VL CDRY (where Y= 1,2, or 3) are from scFvs with
the
same specificity (i.e., from scFvs that bind soluble BLyS (SEQ ID NOS:1563 -
1880),
from scFvs that bind membrane-bound BLyS (SEQ ID NOS:1881 - 2128), or from
scFvs
that bind both soluble and membrane-bound BLyS (SEQ m NOS:1 - 1562). In yet
another embodiment, a composition of the present invention comprises one or
more fusion
proteins.
(0256] As discussed in more detail below, a composition of the invention may
be used
either alone or in combination with other compositions. The antibodies
(including scFvs
and other molecules comprising, or alternatively consisting of antibody
fragments or
variants of the present invention) may further be recombinantly fused to a
heterologous
polypeptide at the N- or C-terminus or chemically conjugated (including
covalently and
non-covalently conjugations) to polypeptides or other compositions. For
example,
antibodies of the present invention may be recombinantly fused or conjugated
to
molecules useful as labels in detection assays and effector molecules such as
heterologous
polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO
92/08495;
WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
[0257] Antibodies of the present invention (including scFvs and other
molecules
comprising, or alternatively consisting of antibody fragments or variants of
the present
invention) may be used, for example, but not limited to, to purify and detect
BLyS, and to
target the polypeptides of the present invention to cells expressing membrane-
bound BLyS
or BLyS receptor, including both in vitro and ira vivo diagnostic and
therapeutic methods.
For example, the antibodies have use in immunoassays for qualitatively and
quantitatively
measuring levels of BLyS in biological samples. See, e.g., Harlow et al.,
Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)
(incorporated
by reference herein in its entirety).
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Methods Producing Antibodies
[0258] The antibodies of the invention (including scFvs and other molecules
comprising, or alternatively consisting of antibody fragments or variants of
the invention)
can be produced by any method known in the art for the synthesis of
antibodies, in
particular, by chemical synthesis or preferably, by recombinant expression
techniques.
[0259] The single chain Fvs disclosed in Table 1 were generated using phage
display
methods known in the art. Furthermore, other scFvs that immunospecifically
bind BLyS
may be generated using phage display methods known in the art. In phage
display
methods, functional antibody domains are displayed on the surface of phage
particles
which carry the polynucleotide sequences encoding them. In particular, DNA
sequences
encoding VH and VL domains are amplified from animal cDNA libraries (e.g.,
human or
murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The
DNA
encoding the VH and VL domains are joined together by an scFv linker by PCR
and
cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is
electroporated in E. coli and the E. coli is infected with helper phage. Phage
used in these
methods are typically filamentous phage including fd and M13 and the VH and VL
domains are usually recombinantly fused to either the phage gene III or gene
VIII. Phage
expressing an antigen binding domain that binds to an antigen of interest
(a.e., BLyS or a
fragment thereof) can be selected or identified with antigen, e.g., using
labeled antigen or
antigen bound or captured to a solid surface or bead. Examples of phage
display methods
that can be used to make the antibodies of the present invention include, but
are not
limited to, those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995);
Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al.,
Eur. J.
Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et
al., Advances
in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01 134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236;
WO 95/15982; WO 95/20401; W097/13844; and U.S. Patent Nos. 5,698,426;
5,223,409;
5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated
herein by
reference in its entirety.
[0260] ScFvs that immunospecifcally bind to both BLyS and APRIL polypeptides
(preferably to the mature soluble forms of each) may be obtained, for example,
by
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sequential rounds of selection for binding to one or the other of BLyS and
APRIL
polypeptides. Thus in one embodiment, the present invention provides for a
method of
selecting phage that express scFvs that immunospecifically bind both BLyS
polypeptides
and APRIL polypeptides, comprising at least one round of phage selection for
binding to
BLyS polypeptide and at least one round of phage selection for binding to
APRIL
polypeptide. Selection for BLyS binding may either precede or follow selection
for
APRIL binding. More than one round of selection for binding to either BLyS or
APRIL
may be conducted.
[0261] ScFvs that immunospecifcally bind to a heterotrimer comprising at least
one
BLyS polypeptide and at least one APRIL polypeptide may be, for example,
obtained by
sequential rounds of selection for binding to one or the other of BLyS and
APRIL
polypeptide. Thus in one embodiment, the present invention provides for a
method of
selecting phage that express scFvs that immunospecifically bind a heterotrimer
comprising
at least one BLyS polypeptide and at least one APRIL polypeptide, comprising
at least one
round of phage selection for binding to BLyS polypeptide and at least one
round of phage
selection for binding to APRIL polypeptide. Selection for BLyS binding may
either
precede or follow selection for APR1L binding. More than one round of
selection for
binding to either BLyS or APRIL may be conducted.
[0262] Alternatively, scFvs that immunospecifcally bind to a heterotrimer
comprising
at least one BLyS polypeptide and at least one APRIL polypeptide may be
obtained, for
example, by selecting scFVs that bind to a BLyS heterotrimer. A BLyS
heterotrimer may
contain, for example, one BLyS polypeptide and two APRIL polypeptides
(2BLyS:lAPRIL heterotrimer). Other BLyS heterotrimers may contain, for
example, one
BLyS polypeptide and two APRIL polypeptides (lBLyS:2APRIL heterotrimer).
Preferably, the heterotrimers comprising BLyS and APRIL polypeptides contain
the
mature forms of both the BLyS and APRIL polypeptides. ScFvs may be selected in
one or
more rounds of selection for binding only to the 2BLyS:lAPRIL heterotrimer, or
only to
the lBLyS:2APRIL heterotrimer. Alternatively, scFVs that immunospecifcally
bind
heterotrimers comprising BLyS and APRIL polypeptides may be obtained through
sequential rounds of screening on individual forms of the BLyS/APRIL
heterotrimer in
any order and/or on both forms of the heterotrimer simultaneously, or any
combination
thereof.
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[0263] As described in the above references, after phage selection, the
antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed
in any desired host, including mammalian cells, insect cells, plant cells,
yeast, and
bacteria, e.g., as described below. Techniques to recombinantly produce Fab,
Fab' and
F(ab')2 fragments can also be employed using methods known in the art such as
those
disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques
12(6):864-
869 (1992); Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science
240:1041-1043
(1988) (said references incorporated by reference in their entireties).
[0264] The characteristics of an antibody, such as its on-rate, off-rate
and/or overall
affinity may be altered using in vitro mutation and selection techniques. This
process is
well known in the art and is commonly referred to as in vitro affinity
maturation of
antibodies. Starting with a given antibody that binds a particular antigen
with a certain
affinity, one of skill in the art can engineer variants of that antibody and
test the antibody
variants for altered (usually improved) antigen binding characteristics. The
amino acid
sequence of the VH and VL regions of such antibody variants may be
substantially from
that of the starting or original antibody; for example, the an antibody
variants may
comprise the original VH or VL paired with a different (from the original) VL
or VH
region, respectively. Alternatively, the amino acid sequence of the VH and VL
region of
such antibody variants may be quite similar to that of the starting or
original antibody; for
example, an antibody variant may have only a few amino acid changes in the VH
and/or
VL region. It is common for one to engineer the mutations in CDR regions, and
particularly in theVHCDR3 region. Examples of both types of in vitro antibody
affinity
maturation are described for example, Thompson et al., (1996) The Journal of
Molecular
Biology 256:77-88. Moreover, a review of phage display antibody technology may
be
found in Vaughan et al., (1998) Nature Biotechnology 16:535-539; both of these
articles
are herein incorporated by reference in their entireties. The scFvs of SEQ ID
NOS:10-37
are CDR3 mutants derived from the scFv of SEQ ID N0:9 and scFvs of SEQ ID
NOS:291-327 are CDR3 mutants derived from the scFv of SEQ m N0:2.
[0265] To generate whole antibodies, PCR primers including VH or VL nucleotide
sequences, a restriction site, and a flanking sequence to protect the
restriction site can be
used to amplify the VH or VL sequences in scFv clones. Utilizing cloning
techniques
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known to those of skill in the art, the PCR amplified VH domains can be cloned
into
vectors expressing a VH constant region, e.g., the human gamma 4 constant
region, and
the PCR amplified VL domains can be cloned into vectors expressing a VL
constant
region, e.g., human kappa or lambda constant regions. Preferably, the vectors
for
expressing the VH or VL domains comprise a promoter suitable to direct
expression of the
heavy and light chains in the chosen expression system, a secretion signal, a
cloning site
for the immunoglobulin variable domain, immunoglobulin constant domains, and a
selection marker such as neomycin. The VH and VL domains may also be cloned
into one
vector expressing the necessary constant regions. The heavy chain conversion
vectors and
light chain conversion vectors are then co-transfected into cell lines to
generate stable or
transient cell lines that express full-length antibodies, e.g., IgG, using
techniques known to
those of skill in the art.
[0266] Cell lines that express antibodies that comprise the VH and VL domains
of
scFvs of the invention have been deposited with the American Type Culture
Collection
("ATCC .") on the dates listed in Table 2 and given the ATCC Deposit Numbers
identified
in Table 2. The ATCC is located at 10801 University Boulevard, Manassas, VA
20110-
2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest
Treaty on
the international recognition of the deposit of microorganisms for purposes of
patent
procedure.
Table 2
Cell Line CorrespondingSEQ ID ATCC DepositATCC Deposit
scFv NO: Number Date
NSO-B11-15 I050B11-15 24 PTA-3238 March 27,
2001
NSO-anti-BLyS-6D08-18I006D08 2 PTA-3239 March 27,
2001
NSO- anti-BLyS-116A01-60I116A01 327 PTA-3240 March 27,
2001
I026C04I~ I026C04-K 1563 PTA-3241 March 27,
2001
I050A12 I050A12 12 PTA-3242 March 27,
2001
I050-B 11 I050B 11 9 PTA-3243 March 27,
2001
[0267] Accordingly, in one embodiment, the invention provides antibodies that
comprise the VH and VL domains of scFvs of the invention.
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[0268] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line NSO-B11-15.
[0269] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line NSO-anti-BLyS-6D08-18.
[0270] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line NSO- anti-BLyS-116A01-60.
[0271] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line I026C04K.
[0272] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line I050A12.
[0273] In a preferred embodiment, an antibody of the invention is the antibody
expressed by cell line NSO-B 11.
[0274] In other preferred embodiments, the invention provides antibodies that
competitively inhibit binding of an antibody comprising a fragment (e.g., VH
domain, VL
domain, VHCDRl, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of
an scFv referred to in Table 1 to a BLyS polypeptide. In preferred
embodiments, the
invention provides antibodies that which reduce the binding of an antibody
comprising a
fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1,
VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a BLyS
polypeptide
by between 1% and 10% in a competitive inhibition assay. In preferred
embodiments, the
invention provides antibodies that which reduce the binding of an antibody
comprising a
fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1,
VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a BLyS
polypeptide
by between 1% and 10% in a competitive inhibition assay.
[0275] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDR1, VHCDR2, VHCDR3, VLCDRl, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 10% and up to 20% in
a
competitive inhibition assay.
[0276] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv
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referred to in Table 1 to a BLyS polypeptide by at least 20% and up to 30% in
a
competitive inhibition assay.
[0277] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDRl, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 30% and up to 40% in
a
competitive inhibition assay.
[0278] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 40% and up to 50% in
a
competitive inhibition assay.
[0279] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 50% and up to 60% in
a
competitive inhibition assay.
[0280] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDRl, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 60% and up to 70% in
a
competitive inhibition assay.
[0281] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDR1, VHCDR2, VHCDR3, VLCDRl, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 70% and up to 80% in
a
competitive inhibition assay.
[0282] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 80% and up to 90% in
a
competitive inhibition assay.
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[0283] In preferred embodiments, the invention provides antibodies that which
reduce
the binding of an antibody comprising a fragment (e.g., VH domain, VL domain,
VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv
referred to in Table 1 to a BLyS polypeptide by at least 90% and up to 100% in
a
competitive inhibition assay.
[0284] In other preferred embodiments, the invention provides antibodies that
competitively inhibit binding of the antibody produced by the cell line having
ATCC
deposit number PTA-3238 to a BLyS polypeptide.
[0285] In other preferred embodiments, the invention provides antibodies that
competitively inhibit binding of the antibody produced by the cell line having
ATCC
deposit number PTA-3239 to a BLyS polypeptide.
[0286] In other preferred embodiments, the invention provides antibodies that
competitively inhibit binding of the antibody produced by the cell line having
ATCC
deposit number PTA-3240 to a BLyS polypeptide.
[0287] In other preferred embodiments, the invention provides antibodies that
competitively inhibit binding of the antibody produced by the cell line having
ATCC
deposit number PTA-3241 to a BLyS polypeptide.
[0288] In other preferred embodiments, the invention provides antibodies that
competitively inhibit binding of the antibody produced by the cell line having
ATCC
deposit number PTA-3242 to a BLyS polypeptide.
[0289] In other preferred embodiments, the invention provides antibodies that
competitively inhibit binding of the antibody produced by the cell line having
ATCC
deposit number PTA-3243 to a BLyS polypeptide.
[0290] For some uses, including if2 vivo use of antibodies in humans and in
vitro
detection assays, it may be preferable to use human or chimeric antibodies.
Completely
human antibodies are particularly desirable for therapeutic treatment of human
patients.
See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO
98/46645,
WO 98/50433, WO 98124893, WO98/16654, WO 96/34096, WO 96/33735, and WO
91/10741; each of which is incorporated herein by reference in its entirety.
In a specific
embodiment, antibodies of the present invention comprise one or more VH and VL
domains corresponding to the human scFvs of the invention and framework
regions from
another imrnunoglobulin molecule, preferably a human immunoglobulin molecule.
In a
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specific embodiment, antibodies of the present invention comprise one or more
CDRs
corresponding to the human scFvs of the invention and framework regions from
another
immunoglobulin molecule, preferably ~a human immunoglobulin molecule. In other
embodiments, an antibody of the present invention comprises one, two, three,
four, five,
six or more VL CDRs or VH CDRs corresponding to one or more of the human scFvs
referred to in Table 1, or fragments or variants thereof, and framework
regions (and,
optionally CDRs not derived from the scFvs in Table 1) from a human
immunoglobulin
molecule. In a preferred embodiment, an antibody of the present invention
comprises a
VH CDR3, VL CDR3, or both, corresponding to the same scFv, or different scFvs
referred
to in Table 1, or fragments or variants thereof, and framework regions from a
human
immunoglobulin.
[0291] A chimeric antibody is a molecule in which different portions of the
antibody
are derived from different immunoglobulin molecules such as antibodies having
a variable
region derived from a human antibody and a non-human immunoglobulin constant
region.
Methods for producing chimeric antibodies are known in the art. See e.g.,
Morrison,
Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Dillies et
al., J.
Immunol. Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715; 4,816,567;
and
4,816,397, which are incorporated herein by reference in their entirety.
Chimeric
antibodies comprising one or more CDRs from human species and framework
regions
from a non-human immunoglobulin molecule (e.g., framework regions from a
canine or
feline immunoglobulin molecule) can be produced using a variety of techniques
known in
the art including, for example, CDR-grafting (EP 239,400; PCT publication WO
91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-
498
(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et
al., PNAS
91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332). In a
preferred
embodiment, chimeric antibodies comprise a human CDR3 having an amino acid
sequence of any one of the VH CDR3s or VL CDR3s referred to in Table 1, or a
variant
thereof, and non-human framework regions or human framework regions different
from
those of the frameworks in the corresponding scFv disclosed in Table 1. Often,
framework residues in the framework regions will be substituted with the
corresponding
residue from the CDR donor antibody to alter, preferably improve, antigen
binding. These
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framework substitutions are identified by methods well known in the art, e.g.,
by modeling
of the interactions of the CDR and framework residues to identify framework
residues
important for antigen binding and sequence comparison to identify unusual
framework
residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated herein by
reference in
their entireties.)
[0292] Further, the antibodies of the invention can, in turn, be utilized to
generate anti-
idiotype antibodies that "mimic" BLyS polypeptides using techniques well known
to those
skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444
(1993); and
Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies of
the
invention which bind to BLyS and competitively inhibit the binding of BLyS to
its
receptor (as determined by assays well known in the art such as, for example,
that
disclosed, infra) can be used to generate antiidiotypes that "mimic" a BLyS
ligand/receptor-binding domain and, as a consequence, bind to and neutralize
BLyS
receptors (e.g., TACI - GenBank accession number AAC51790; BCMA - GenBank
accession number NP_001183; and/or BAFF-R - GenBank accession number
NP 443177). Such neutralizing anti-idiotypes (including molecules comprising,
or
alternatively consisting of, antibody fragments or variants, such as Fab
fragments of such
anti-idiotypes) can be used in therapeutic regimens to neutralize BLyS. For
example, such
anti-idiotypic antibodies can be used to bind BLyS ligands/receptors, and
thereby block
BLyS mediated biological activity. Alternatively, anti-idiotypes that "mimic"
a BLyS
binding domain may bind to BLyS receptors) and induce BLyS receptor mediated
signalling (e.g., activation of nuclear factor of activated T cells (NF-AT),
nuclear factor-
kappa B (NF-kappa B), and/or AP-1). Such agonistic anti-idiotypes (including
agonistic
Fab fragments of these anti-idiotypes) can be used in therapeutic regimens to
induce or
enhance BLyS receptor mediated signalling. For example, such anti-idiotypic
antibodies
can be used to bind BLyS ligands/receptors, and thereby stimulate BLyS
mediated
biological activity (e.g., B cell proliferation and/or immunoglobulin
production).
(0293] Once an antibody molecule of the invention (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) has
been chemically
synthesized or recombinantly expressed, it may be purified by any method known
in the
art for purification of an immunoglobulin molecule, or more generally, a
protein molecule,
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such as, for example, by chromatography (e.g., ion exchange, affinity,
particularly by
affinity for the specific antigen after Protein A, and sizing column
chromatography),
centrifugation, differential solubility, or by any other standard technique
for the
purification of proteins. Further, the antibodies of the present invention may
be fused to
heterologous polypeptide sequences described herein or otherwise known in the
art, to
facilitate purification.
Polynucleotides Encoding an Antibody
[0294] The invention provides polynucleotides comprising, or alternatively
consisting
of, a nucleotide sequence encoding an antibody of the invention (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof). The
invention also encompasses polynucleotides that hybridize under high
stringency, or
alternatively, under intermediate or lower stringency hybridization
conditions, e.g., as
defined supra, to polynucleotides complementary to nucleic acids having a
polynucleotide
sequence that encodes an antibody of the invention or a fragment or variant
thereof.
[0295] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. Since the amino
acid
sequences of the scFv antibodies and VH domains, VL domains and CDRs thereof,
are
known (as described in Table 1), nucleotide sequences encoding these
antibodies can be
determined using methods well known in the art, i.e., the nucleotide codons
known to
encode the particular amino acids are assembled in such a way to generate a
nucleic acid
that encodes the antibody, of the invention. Such a polynucleotide encoding
the antibody
may be assembled from chemically synthesized oligonucleotides (e.g., as
described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the
synthesis of
overlapping oligonucleotides containing portions of the sequence encoding the
antibody,
annealing and ligating of those oligonucleotides, and then amplification of
the ligated
oligonucleotides by PCR.
[0296] Alternatively, a polynucleotide encoding an antibody (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) may be
generated from nucleic acid from a suitable source. If a clone containing a
nucleic acid
encoding a particular antibody is not available,,but the sequence of the
antibody molecule
is known, a nucleic acid encoding the immunoglobulin may be chemically
synthesized or
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obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA
library
generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any
tissue or
cells expressing the antibody, such as hybridoma cells selected to express an
antibody of
the invention) by PCR amplification using synthetic primers hybridizable to
the 3' and 5'
ends of the sequence or by cloning using an oligonucleotide probe specific for
the
particular gene sequence to identify, e.g., a cDNA clone from a cDNA library
that encodes
the antibody. Amplified nucleic acids generated by PCR may then be cloned into
replicable cloning vectors using any method well known in the art.
[0297] Once the nucleotide sequence of the antibody (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) is
determined, the
nucleotide sequence of the antibody may be manipulated using methods well
known in the
art for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site
directed mutagenesis, PCR, etc. (see, for example, the techniques described in
Sambrook
et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor
Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current
Protocols in
Molecular Biology, John Wiley & Sons, NY, which are both incorporated by
reference
herein in their entireties), to generate antibodies having a different amino
acid sequence,
for example to create amino acid substitutions, deletions, and/or insertions.
[0298] In a specific embodiment, one or more of the VH and VL domains referred
to
in Table l, or fragments or variants thereof, is inserted within framework
regions using
recombinant DNA techniques known in the art. In a specific embodiment, one,
two, three,
four, five, six, or more of the CDRs referred to in Table 1, or fragments or
variants
thereof, is inserted within framework regions using recombinant DNA techniques
known
in the art. The framework regions may be naturally occurring or consensus
framework
regions, and preferably human framework regions (see, e.g., Chothia et al., J.
Mol. Biol.
278: 457-479 (1998) for a listing of human framework regions, the contents of
which are
hereby incorporated by reference in its entirety). Preferably, the
polynucleotides
generated by the combination of the framework regions and CDRs encode an
antibody
(including molecules comprising, or alternatively consisting of, antibody
fragments or
variants thereof) that specifically binds to BLyS. Preferably, as discussed
supra,
polynucleotides encoding variants of antibodies or antibody fragments having
one or more
amino acid substitutions may be made within the framework regions, and,
preferably, the
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amino acid substitutions improve binding of the antibody to its antigen.
Additionally,
such methods may be used to make amino acid substitutions or deletions of one
or more
variable region cysteine residues participating in an intrachain disulfide
bond to generate
antibody molecules, or antibody fragments or variants, lacking one or more
intrachain
disulfide bonds. Other alterations to the polynucleotide are encompassed by
the present
invention and fall within the ordinary skill of the art.
Recombinant Expression of an Antibody
[0299] Recombinant expression of an antibody of the invention (including scFvs
and
other molecules comprising, or alternatively consisting of, antibody fragments
or variants
thereof (e.g., a heavy or light chain of an antibody of the invention or a
portion thereof or
a single chain antibody of the invention)), requires construction of an
expression vectors)
containing a polynucleotide that encodes the antibody. Once a polynucleotide
encoding
an antibody molecule (e.g., a whole antibody, a heavy or light chain of an
antibody, or
portion thereof (preferably, but not necessarily, containing the heavy or
light chain
variable domain)), of the invention has been obtained, the vectors) for the
production of
the antibody molecule may be produced by recombinant DNA technology using
techniques well known in the art. Thus, methods for preparing a protein by
expressing a
polynucleotide containing an antibody encoding nucleotide sequence are
described herein.
Methods which are well known to those skilled in the art can be used to
construct
expression vectors containing antibody coding sequences and appropriate
transcriptional
and translational control signals. These methods include, for example, ifa
vitro
recombinant DNA techniques, synthetic techniques, and in vivo genetic
recombination.
The invention, thus, provides replicable vectors comprising a nucleotide
sequence
encoding an antibody molecule of the invention (e.g., a whole antibody, a
heavy or light
chain of an antibody, a heavy or light chain variable domain of an antibody,
or a portion
thereof, or a heavy or light chain CDR, a single chain Fv, or fragments or
variants
thereof), operably linked to a promoter. Such vectors may include the
nucleotide
sequence encoding the constant region of the antibody molecule (see, e.g., PCT
Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No.
5,122,464, the contents of each of which are hereby incorporated by reference
in its
entirety) and the variable domain of the antibody may be cloned into such a
vector for
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expression of the entire heavy chain, the entire light chain, or both the
entire heavy and
light chains.
[0300] The expression vectors) is(are) transferred to a host cell by
conventional
techniques and the transfected cells are then cultured by conventional
techniques to
produce an antibody of the invention. Thus, the invention includes host cells
containing
polynucleotide(s) encoding an antibody of the invention (e.g., whole antibody,
a heavy or
light chain thereof, or portion thereof, or a single chain antibody of the
invention, or a
fragment or variant thereof), operably linked to a heterologous promoter. In
preferred
embodiments, for the expression of entire antibody molecules, vectors encoding
both the
heavy and light chains may be co-expressed in the host cell for expression of
the entire
immunoglobulin molecule, as detailed below.
[0301] A variety of host-expression vector systems may be utilized to express
the
' antibody molecules of the invention. Such host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but
also represent cells which may, when transformed or transfected with the
appropriate
nucleotide coding sequences, express an antibody molecule of the invention in
situ. These
include, but are not limited to, microorganisms such as bacteria (e.g., E.
coli, B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA
expression vectors containing antibody coding sequences; yeast (e.g.,
Saccharofnyees,
Pichia) transformed with recombinant yeast expression vectors containing
antibody
coding sequences; insect cell systems infected with recombinant virus
expression vectors
(e.g., baculovirus) containing antibody coding sequences; plant cell systems
infected with
recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco
mosaic virus, TMV) or transformed with recombinant plasmid expression vectors
(e.g., Ti
plasmid) containing antibody coding sequences; or mammalian cell systems
(e.g., COS,
CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs
containing
promoters derived from the genome of mammalian cells (e.g., metallothionein
promoter)
or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia
virus 7.5K
promoter). Preferably, bacterial cells such as Escherichia coli, and more
preferably,
eukaryotic cells, especially for the expression of whole recombinant antibody
molecule,
are used for the expression of a recombinant antibody molecule. For example,
mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with
a vector
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such as the major intermediate early gene promoter element from human
cytomegalovirus
is an effective expression system for antibodies (Foecking et al., Gene 45:101
(1986);
Cockett et al., Bio/Technology 8:2 (1990)).
[0302] In bacterial systems, a number of expression vectors may be
advantageously
selected depending upon the use intended for the antibody molecule being
expressed. For
example, when a large quantity of such a protein is to be produced, for the
generation of
pharmaceutical compositions of an antibody molecule, vectors which direct the
expression
of high levels of fusion protein products that are readily purified may be
desirable. Such
vectors include, but are not limited to, the E. coli expression vector pUR278
(Ruther et al.,
EMBO 1. 2:1791 (1983)), in which the antibody coding sequence may be ligated
individually into the vector in frame with the lac Z coding region so that a
fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109
(1985); Van
Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors may
also be used to express foreign polypeptides as fusion proteins with
glutathione 5-
transferase (GST). In general, such fusion proteins are soluble and can easily
be purified
from lysed cells by adsorption and binding to matrix glutathione agarose beads
followed
by elution in the presence of free glutathione. The pGEX vectors are designed
to include
thrombin or factor Xa protease cleavage sites so that the cloned target gene
product can be
released from the GST moiety.
[0303] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) may be used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. Antibody coding sequences may be cloned
individually into
non-essential regions (for example, the polyhedrin gene) of the virus and
placed under
control of an AcNPV promoter (for example, the polyhedrin promoter).
[0304] In mammalian host cells, a number of viral-based expression systems may
be
utilized. In cases where an adenovirus is used as an expression vector, the
antibody
coding sequence of interest may be ligated to an adenovirus
transcription/translation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric gene
may then be inserted in the adenovirus genome by ifz vitro or ih vivo
recombination.
Insertion in a non-essential region of the viral genome (e.g., region El or
E3) will result in
a recombinant virus that is viable and capable of expressing the antibody
molecule in
infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-
359 (1984)).
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Specific initiation signals may also be required for efficient translation of
inserted
antibody coding sequences. These signals include the ATG initiation.codon and
adjacent
sequences. Furthermore, the initiation codon must be in phase with the reading
frame of
the desired coding sequence to ensure translation of the entire insert. These
exogenous
translational control signals and initiation codons can be of a variety of
origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of
appropriate transcription enhancer elements, transcription terminators, etc.
(see, e.g.,
Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
[0305] In addition, a host cell strain may be chosen which modulates the
expression of
the inserted sequences, or modifies and processes the gene product in the
specific fashion
desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein
products may be important for the function of the protein. Different host
cells have
characteristic and specific mechanisms for the post-translational processing
and
modification of proteins and gene products. Appropriate cell lines or host
systems can be
chosen to ensure the correct modification and processing of the foreign
protein expressed.
To, this end, eukaryotic host cells which possess the cellular machinery for
proper
processing of the primary transcript, glycosylation, and phosphorylation of
the gene
product may be used. Such mammalian host cells include, but are not limited
to, CHO,
VERY, BHK, Hela, COS, NSO, MDCK, 293, 3T3, W138, and in particular, breast
cancer
cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal
mammary gland cell line such as, for example, CRL7O3O and HsS78Bst.
[0306] For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody may be
engineered. Rather than using expression vectors which contain viral origins
of
replication, host cells can be transformed with DNA controlled by appropriate
expression
control elements (e.g., promoter, enhancer, sequences, transcription
terminators,
polyadenylation sites, etc.), and a selectable marker. Following the
introduction of the
foreign DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media,
and then are switched to a selective media. The selectable marker in the
recombinant
plasmid confers resistance to the selection and allows cells to stably
integrate the plasmid
into their chromosomes and grow to form foci which in turn can be cloned and
expanded
into cell lines. This method may advantageously be used to engineer cell lines
which
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express the antibody molecule. Such engineered cell lines may be particularly
useful in
screening and evaluation of compositions that interact directly or indirectly
with the
antibody molecule.
[0307] A number of selection systems may be used, including but not limited
to, the
herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthine-
guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad.
Sci. USA
48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8
17 (1980))
genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also,
antimetabolite
resistance can be used as the basis of selection for the following genes:
dhfY, which
confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357
(1980);
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance
to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072
(1981));
neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy
12:488-
505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol.
32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and
Anderson,
Ann. Rev. Biochem. 62: 191-217 (1993); TIB TECH 11(5):155-2 15 (May, 1993));
and
hygro, which /confers resistance to hygromycin (Santerre et al., Gene 30:147
(1984)).
Methods commonly known in the art of recombinant DNA technology may be
routinely
applied to select the desired recombinant clone, and such methods are
described, for
example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology,
John Wiley &
Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds),
Current
Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et
al., J.
Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their
entireties.
[0308] The expression levels of an antibody molecule can be increased by
vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on
gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system
expressing antibody is amplifiable, increase in the level of inhibitor present
in culture of
host cell will increase the number of copies of the marker gene. Since the
amplified
region is associated with the coding sequence of the antibody, production of
the antibody
will also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
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[0309] The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second
vector encoding a light chain derived polypeptide. The two vectors may contain
identical
selectable markers which enable equal expression of heavy and light chain
polypeptides.
Alternatively, a single vector may be used which encodes, and is capable of
expressing,
both heavy and light chain polypeptides. In such situations, the light chain
is preferably
placed before the heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot,
Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2 197 (1980)). The
coding
sequences for the heavy and light chains may comprise cDNA or genomic DNA.
[0310] Once an antibody molecule of the invention has been produced by
recombinant
expression, it may be purified by any method known in the art for purification
of an
immunoglobulin molecule, or more generally, for purification of a protein, for
example,
by chromatography (e.g., ion exchange, affinity, particularly by affinity for
the specific
antigen after Protein A, and sizing column chromatography), centrifugation,
differential
solubility, or by any other standard technique for the purification of
proteins. Further, the
antibodies of the present invention may be fused to heterologous polypeptide
sequences
described herein or otherwise known in the art to facilitate purification.
[0311] Antibodies of the present invention include naturally purified
products,
products of chemical synthetic procedures, and products produced by
recombinant
techniques from a prokaryotic or eukaryotic host, including, for example,
bacterial, yeast,
higher plant, insect and mammalian cells. Depending upon the host 'employed in
a
recombinant production procedure, the antibodies of the present invention may
be
glycosylated or may be non-glycosylated. In addition, antibodies of the
invention may also
include an initial modified methionine residue, in some cases as a result of
host-mediated
processes.
[0312] Antibodies of the invention can be chemically synthesized using
techniques
known in the art (e.g., see Creighton, 1983, Proteins: Structures and
Molecular Principles,
W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310:105-
111). For
example, a peptide corresponding to a fragment of an antibody of the invention
can be
synthesized by use of a peptide synthesizer. Furthermore, if desired,
nonclassical amino
acids or chemical amino acid analogs can be introduced as a substitution or
addition into
the antibody polypeptide sequence. Non-classical amino acids include, but are
not limited
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to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-
amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,
6-amino
hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,
ornithine,
norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,
cysteic acid, t-
butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,
fluoro-amino
acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino
acids, Na-
methyl amino acids, and amino acid analogs in general. Furthermore, the amino
acid can
be D (dextrorotary) or L (levorotary).
[0313] The invention encompasses antibodies which are differentially modified
during
or after translation, e.g., by glycosylation, acetylation, phosphorylation,
amidation,
derivatization by known protecting/blocking groups, proteolytic cleavage,
linkage to an
antibody molecule or other cellular ligand, etc. Any of numerous chemical
modifications
may be carried out by known techniques, including but not limited, to specific
chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
NaBH4.,
acetylation, formylation, oxidation, reduction, metabolic synthesis in the
presence of
tunicamycin, etc.
[0314] Additional post-translational modifications encompassed by the
invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic host cell
expression. The polypeptides may also be modified with a detectable label,
such as an
enzymatic, fluorescent, radioisotopic or affinity label to allow for detection
and isolation
of the antibody.
[0315] Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, glucose oxidase or acetylcholinesterase;
examples of
suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin;
examples of suitable fluorescent materials include biotin, umbelliferone,
fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride
or phycoerythrin; an example of a luminescent material includes luminol;
examples of
bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of
suitable radioactive material include a radioactive metal ion, e.g., alpha-
emitters such as,
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for example, 21381, or other radioisotopes such as, for example, iodine (1311,
lash 1231, 121I),
carbon (14C), sulfur (35S), tritium (3IT), indium (lls~In, 113mIn, llz~, 1110,
and technetium
(~9Tc, 99mT'c), thallium (2olTi), gallium (68Ga, 67Ga), palladium (lo3Pd),
molybdenum
(99M0), xenon (133~e), ~uorlne (18F), 153Sm, 177Lu, 159Gd, 149Pm~ 140La, 17s~,
166H~~ 90Y,
47s~, 186Re, 188Re, 142Pr, 105Rh, 97Ru~ 68Ge, 57C~, 65zn, 85sr, 32P, 153Gd,
169, Sl~r, 54~
7sSe,113Sn, and 117Tin.
[0316] In specific embodiments, antibodies of the invention may be labeled
with
Europium. For example, antibodies of the invention may be labelled with
Europium using
the DELFIA Eu-labeling kit (catalog# 1244-302, Perkin Elmer Life Sciences,
Boston,
MA) following manufacturer's instructions.
[0317] In specific embodiments, antibodies of the invention are attached to
macrocyclic chelators useful for conjugating radiometal ions, including but
not limited to,
111, 177Lu, 9oY, 166Ho, and ls3Sm, to antibodies. In a preferred embodiment,
the
radiometal ion associated with the macrocyclic chelators attached to
antibodies of the
invention is 111In. In another preferred embodiment, the radiometal ion
associated with
the macrocyclic chelator attached to antibodies of the invention is
~°Y. In specific
embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-
N,N',N",N"'-
tetraacetic acid (DOTA). In specific embodiments, the macrocyclic chelator is
cc-(5-
isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraaza- cyclododecane-1,4,7,10-
tetraacetic
acid. In other specific embodiments, the DOTA is attached to the antiboddy of
the
invention via a linker molecule. Examples of linker molecules useful for
conjugating
DOTA to an antibody are commonly known in the art - see, for example, DeNardo
et al.,
Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem.
10(4):553-7,
1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are
hereby
incorporated by reference in their entirety. In addition, U.S. Patents
5,652,361 and
5,756,065, which disclose chelating agents that may be conjugated to
antibodies, and
methods for making and using them, are hereby incorporated by reference in
their
entireties.
[031] In one embodiment, antibodies of the invention are labeled with biotin.
In
other related embodiments, biotinylated antibodies of the invention may be
used, for
example, as an imaging agent or as a means of identifying one or more BLyS
receptors)
or other coreceptor or ligand molecules.
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[0319] Also provided by the invention are chemically modified derivatives of
antibodies of the invention which may provide additional advantages such as
increased
solubility, stability and in vivo or in vitro circulating time of the
polypeptide, or decreased
immunogenicity (see U. S. Patent No. 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene glycol,
ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran,
polyvinyl
alcohol and the like. The polypeptides may be modified at random positions
within the
molecule, or at predetermined positions within the molecule and may include
one, twos
three or more attached chemical moieties.
[0320] The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1
kDa and about 100 kDa (the term "about" indicating that in preparations of
polyethylene
glycol, some molecules will weigh more, some less, than the stated molecular
weight) for
ease in handling and manufacturing. Other sizes may be used, depending on the
desired
therapeutic profile (e.g., the duration of sustained release desired, the
effects, if any. on
biological activity, the ease in handling, the degree or lack of antigenicity
and other known
effects of the polyethylene glycol to a therapeutic protein or analog). For
example, the
polyethylene glycol may have an average molecular weight of about 200, 500,
1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500,
14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500,
19,000, 19,500,
20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000,
70,000, 75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kI~a.
[0321] As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575;
Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al.,
Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Biocorzjug.
Chem.
10:638-646 (1999), the disclosures of each of which are incorporated herein by
reference.
[0322] The polyethylene glycol molecules (or other chemical moieties) should
be
attached to the protein with consideration of effects on functional or
antigenic domains of
the antibody. There are a number of attachment methods available to those
skilled in the
art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-
CSF), see
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also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of
GM-CSF
using tresyl chloride). For example, polyethylene glycol may be covalently
bound through
amino acid residues via a reactive group, such as, a free amino or carboxyl
group.
Reactive groups are those to which an activated polyethylene glycol molecule
may be
bound. The amino acid residues having a free amino group may include, for
example,
lysine residues and the N-terminal amino acid residues; those having a free
carboxyl group
may include aspartic acid residues, glutamic acid residues, and the C-terminal
amino acid
residue. Sulfhydryl groups may also be used as a reactive group for attaching
the
polyethylene glycol molecules. Preferred for therapeutic purposes is
attachment at an
amino group, such as attachment at the N-terminus or lysine group.
[0323] As suggested above, polyethylene glycol may be attached to proteins,
e.g.,
antibodies, via linkage to any of a number of amino acid residues. For
example,
polyethylene glycol can be linked to a proteins via covalent bonds to lysine,
histidine,
aspartic acid, glutamic acid, or cysteine residues. One or more reaction
chemistries may
be employed to attach polyethylene glycol to specific amino acid residues
(e.g., lysine,
histidine, aspartic acid, glutamic acid, or cysteine) of the antibody or to
more than one
type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic
acid, cysteine and
combinations thereof) of the antibody.
[0324] One may specifically desire antibodies chemically modified at the N-
terminus
of either the heavy chain or the light chain or both. Using polyethylene
glycol as an
illustration, one may select from a variety of polyethylene glycol molecules
(by molecular
weight, branching, etc.), the proportion of polyethylene glycol molecules to
protein (or
peptide) molecules in the reaction mix, the type of pegylation reaction to be
performed,
and the method of obtaining the selected N-terminally pegylated protein. The
method of
obtaining the N-terminally pegylated preparation (i.e., separating this moiety
from other
monopegylated moieties if necessary) may be by purification of the N-
terminally
pegylated material from a population of pegylated protein molecules. Selective
chemical
modification at the N-terminus may be accomplished by reductive alkylation
which
exploits differential reactivity of different types of primary amino groups
(lysine versus
the N-terminal) available for derivatization in a particular antibody, e.g., a
heavy chain or
alight chain. Under the appropriate reaction conditions, substantially
selective
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derivatization of the protein at the N-terminus with a carbonyl group
containing polymer
is achieved.
[0325] As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may be
attached to the protein either directly or by an intervening linker.
Linkerless systems for
attaching polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Theca.
Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hernatol.
68:1-18 (1998);
U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO
98/32466,
the disclosures of each of which are incorporated herein by reference.
[0326] One system for attaching polyethylene glycol directly to amino acid
residues of
proteins without an intervening linker employs tresylated MPEG, which is
produced by
the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride
(C1S02CH2CF3). Upon reaction of antibody with tresylated MPEG, polyethylene
glycol is
directly attached to amine groups of the antibody. Thus, the invention
includes antibody-
polyethylene glycol conjugates produced by reacting proteins of the invention
with a
polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[0327] Polyethylene glycol can also be attached to antibodies using a number
of
different intervening linkers. For example, U.S. Patent No. 5,612,460, the
entire
disclosure of which is incorporated herein by reference, discloses urethane
linkers for
connecting polyethylene glycol to proteins. Protein-polyethylene glycol
conjugates
wherein the polyethylene glycol is attached to the antibody by a linker can
also be
produced by reaction of antibodies with compounds such as MPEG-
succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-
2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-
succinate derivatives. A number additional polyethylene glycol derivatives and
reaction
chemistries for attaching polyethylene glycol to proteins are described in WO
98/32466,
the entire disclosure of which is incorporated herein by reference. Pegylated
protein
products produced using the reaction chemistries set out herein are included
within the
scope of the invention.
[0328] The number of polyethylene glycol moieties attached to each antibody of
the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated
antibodies of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12, 15,
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17, 20, or more polyethylene glycol molecules. Similarly, the average degree
of
substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-
11, 10-12, 11-
13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol
moieties per
antibody molecule. Methods for determining the degree of substitution are
discussed, for
example, in Delgado et al., Crit. Rev. Tlaera. Drug CaYrier Sys. 9:249-304
(1992).
Antibody Characterization
[0329] Antibodies of the present invention (including scFvs and other
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) may be
characterized in a variety of ways. In particular, antibodies and related
molecules of the
invention may be assayed for the ability to immunospecifically bind to BLyS or
a
fragment of BLyS (e.g., to the soluble form or the membrane-bound form of
BLyS) using
techniques described herein or routinely modifying techniques known in the
art. BLyS or
BLyS fragments that may be immunospecifically bound by the compositions of the
invention include, but are not limited to, human BLyS (SEQ ID NOS:3228 and/or
3229)
or BLyS expressed on human monocytes; murine BLyS (SEQ ID NOS:3230 andlor
3231)
or BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as
given in
SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form,
e.g., on
the surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS
polypeptides of
SEQ m NOS:3236 and/or 3237, the soluble form of monkey BLyS, or BLyS expressed
on
monkey monocytes) or fragments thereof. Preferably compositions of the
invention bind
human BLyS (SEQ ID NOS:3228 and/or 3229) or fragments thereof. Assays for the
ability of the antibodies of the invention to immunospecifically bind BLyS or
a fragment
of BLyS may be performed in solution (e.g., Houghten, Bio/Techniques
13:412-421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips
(e.g., Fodor,
Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Patent No. 5,223,409), on
spores (e.g.,
Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et
al., Proc.
Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith,
Science
249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc.
Natl. Acad.
Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991))
(each of
these references is incorporated herein in its entirety by reference).
Antibodies that have
been identified to immunospecifically bind to BLyS or a fragment of BLyS can
then be
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assayed for their specificity and affinity for BLyS or a fragment of BLyS
using or
routinely modifying techniques described herein or otherwise known in the art.
[0330] The antibodies of the invention may be assayed for immunospecific
binding to
BLyS and cross-reactivity with other antigens by any method known in the art.
In
particular, the ability of an antibody to immunospecifically bind to the
soluble form or
membrane-bound form of BLyS and the specificity of the antibody, fragment, or
variant
for BLyS polypeptide from a particular species (e.g., murine, monkey or human,
preferably human) may be determined using or routinely modifying techniques
described
herein or otherwise known in art .
[0331] Immunoassays which can be used to analyze immunospecific binding and
cross-reactivity include, but are not limited to, competitive and non-
competitive assay
systems using techniques such as western blots, radioimmunoassays, ELISA
(enzyme
linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays,
precipitin reactions, gel diffusion precipitin reactions, immunodiffusion
assays,
agglutination assays, complement-fixation assays, immunoradiometric assays,
fluorescent
immunoassays, and protein A immunoassays, to name but a few. Such assays are
routine
and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is
incorporated by
reference herein in its entirety). Exemplary immunoassays are described
briefly below
(but are not intended by way of limitation).
[0332] Tmmunoprecipitation protocols generally comprise lysing a population of
cells
in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium
deoxycholate, 0.1 % SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1 %
Trasylol)
supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA,
PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the cell
lysate, incubating
for a period of time (e.g., 1 to 4 hours) at 40 degrees C, adding protein A
and/or protein G
sepharose beads to the cell lysate, incubating for about an hour or more at 40
degrees C,
washing the beads in lysis buffer and resuspending the beads in SDS/sample
buffer. The
ability of the antibody of interest to immunoprecipitate a particular antigen
can be
assessed by, e.g., western blot analysis. One of skill in the art would be
knowledgeable as
to the parameters that can be modified to increase the binding of the antibody
to an antigen
and decrease the background (e.g., pre-clearing the cell lysate with sepharose
beads). For
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further discussion regarding immunoprecipitation protocols see, e.g., Ausubel
et al, eds,
1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,
New
York at 10.16.1.
[0333] Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein
sample from
the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon,
blocking the
membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing
the
membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with
primary
antibody (the antibody of interest) diluted in blocking buffer, washing the
membrane in
washing buffer, blocking the membrane with a secondary antibody (which
recognizes the
primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic
substrate
(e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule
(e.g., 32P or
izsl) diluted in blocking buffer, washing the membrane in wash buffer, and
detecting the
presence of the antigen. One of skill in the art would be knowledgeable as to
. the
parameters that can be modified to increase the signal detected and to reduce
the
background noise. For further discussion regarding western blot protocols
see,. e.g.,
Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John
Wiley &
Sons, Inc., New York at 10.8.1.
[0334] ELISAs comprise preparing antigen, coating the well of a 96-well
microtiter
plate with the antigen, washing away antigen that did not bind the wells,
adding the
antibody of interest conjugated to a detectable compound such as an enzymatic
substrate
(e.g., horseradish peroxidase or alkaline phosphatase) to the wells and
incubating for a
period of time, washing away unbound antibodies or non-specifically bound
antibodies,
and detecting the presence of the antibodies specifically bound to the antigen
coating the
well. In ELISAs the antibody of interest does not have to be conjugated to a
detectable
compound; instead, a second antibody (which recognizes the antibody of
interest)
conjugated to a detectable compound may be added to the well. Further, instead
of
coating the well with the antigen, the antibody may be coated to the well. In
this case, the
detectable molecule could be the antigen conjugated to a detectable compound
such as an
enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase).
One of skill in
the art would be knowledgeable as to the parameters that can be modified to
increase the
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signal detected as well as other variations of ELISAs known in the art. For
further
discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in
Molecular Biology, Vol. l, John Wiley ~ Sons, Inc., New York at 11.2.1.
[0335] The binding affinity of an antibody (including an scFv or other
molecule
comprising, or alternatively consisting of, antibody fragments or variants
thereof) to an
antigen and the off-rate of an antibody-antigen interaction can be determined
by
competitive binding assays. One example of a competitive binding assay is a
radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or
lzsl) with the
antibody of interest in the presence of increasing amounts of unlabeled
antigen, and the
detection of the antibody bound to the labeled antigen. The affinity of the
antibody of the
present invention for BLyS and the binding off rates can be determined from
the data by
Scatchard plot analysis. Competition with a second antibody can also be
determined using
radioimmunoassays. In this case, BLyS is incubated with an antibody of the
present
invention conjugated to a labeled compound (e.g., 3H or l2sI) in the presence
of increasing
amounts of an unlabeled second anti-BLyS antibody.
[0336] In a preferred embodiment, BIAcore kinetic analysis is used to
determine the
binding on and off rates of antibodies (including an scFv or other molecule
comprising, or
alternatively consisting of, antibody fragments or variants thereof) to BLyS,
or fragments
of BLyS. BIAcore kinetic analysis comprises analyzing the binding and
dissociation of
BLyS from chips with immobilized antibodies on their surface as described in
detail in
Examples 6, 12, 17 and 18, ifzfra.
[0337] The antibodies of the invention (including scFvs and other molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) can also
be assayed for their ability to inhibit, increase, or not significantly alter,
the binding of
BLyS to a BLyS receptor (e.g., TACI - GenBank accession number AAC51790; BCMA -
GenBank accession number NP_001183; and/or BAFF-R - GenBank accession number
NP 443177) using techniques known to those of skill in the art. For example,
cells
expressing a receptor for BLyS (e.g., IM9, REH, ARH-77cells, Namalwa, and RPMI-
8226
B cell tumor lines as wells as peripheral CD20+ B cells) can be contacted with
BLyS in
the presence or absence of an antibody, and the ability of the antibody to
inhibit, increase,
or not significantly alter, BLyS binding to the cells can be measured. BLyS
binding to
cells can be measured by, for example, flow cytometry or a scintillation
assay. BLyS or
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the antibody can be labeled with a detectable compound such as a radioactive
label (e.g.,
32p, 355 and 125I) or a fluorescent label (e.g., fluorescein isothiocyanate,
rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine)
to
enable detection of an interaction between BLyS and a BLyS receptor and/or
BLyS and an
antibody of the invention. Alternatively, the ability of antibodies of the
invention to
inhibit, increase, or not significantly alter, BLyS binding to a BLyS receptor
can be
determined in cell-free assays. For example, native or recombinant BLyS (e.g.,
that
having the amino acid sequence of amino acids 134 - 285 of SEQ ID N0:3228) or
a
fragment thereof can be contacted with an antibody and the ability of the
antibody to
inhibit, increase, or not significantly alter, BLyS from binding to a BLyS
receptor can be
determined. Preferably, the antibody is immobilized on a solid support and
BLyS or a
BLyS fragment is labeled with a detectable compound. Alternatively, BLyS or a
BLyS
fragment is immobilized on a solid support and the antibody is labeled with a
detectable
compound. BLyS may be partially or completely purified (e.g., partially or
completely
free of other polypeptides) or part of a cell lysate. Further, the BLyS
polypeptide may be
a fusion protein comprising BLyS or a biologically active portion thereof and
a domain
such as an Iminunoglobulin Fc or glutathionine-S-transferase. For example,
amino acid
residues 1-154 of TACI (GenBank accession number AAC51790), or 1-48 of BCMA
(GenBank accession number NP_001183) may be fused to the Fc region of an IgG
molecule and used in a cell free assay to determine the ability of antibodies
of the
invention to inhibit, increase, or not significantly alter, BLyS binding to a
BLyS receptor.
Alternatively, BLyS can be biotinylated using techniques well known to those
of skill in
the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL,).
[0338] The antibodies of the invention (including scFvs or other molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof), can also
be assayed for their ability to inhibit, stimulate, or not significantly
alter, BLyS-induced B-
cell proliferation using techniques known to those of skill in the art. For
example, B-cell
proliferation can be assayed by 3H-thymidine incorporation assays and trypan
blue cell
counts (see, e.g., Moore et al., Science 285: 260-263 (1999)). Further, the
antibodies of
the invention, or fragments or variants thereof, can be assayed for their
ability to block,
stimulate, or not significantly alter, BLyS-induced activation of cellular
signaling
molecules and transcription factors such as calcium-modulator and cyclophilin
ligand
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("CAML"), calcineurin, nuclear factor of activated T cells transcription
factor ("NF-AT"),
nuclear factor-kappa B ("NF-kappa B"), and AP-1 using techniques known to
those of
skill in the art (see, e.g., von Bulow and Bram, Science 278:138-141(1997)).
For
example, NF-AT activity can be determined by electromobility gel shift assays,
by
detecting the expression of a protein known to be regulated by NF-AT (e.g., IL-
2
expression), by detecting the induction of a reporter gene ( e.g., an NF-AT
regulatory
element operably linked to a nucleic acid encoding a detectable marker such as
luciferase,
beta-galactosidase or chloramphenicol acetyltransferase (CAT)), or by
detecting a cellular
response (e.g., cellular differentiation, or cell proliferation).
[0339] The antibodies of the invention, or fragments or variants thereof can
also be
assayed for their ability to neutralize, enhance, or not significantly alter,
BLyS activity.
For example, antibodies or fragments or variants thereof, may be routinely
tested for their
ability to inhibit BLyS from binding to cells expressing the receptor for BLyS
(see
Example 3, infra).
Selection and Screening for Antibodies that Immunospecifically Bind to Soluble
BLyS
[0340] Antibodies of the invention (including scFvs and other molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) may be
screened in a
variety of assays to identify those antibodies that immunospecifically bind to
the soluble
form of BLyS. In one particular assay, antibodies that bind to the
biotinylated soluble
form of BLyS in solution are captured on streptavidin coated magnetic beads.
This assay
may be relatively applied to identify antibodies of the invention that
neutralize and/or bind
to BLyS. Additionally, antibodies may be assayed in neutralization assays
described
herein or otherwise known in the art (see Example 3, infra). For example,
antibodies may
be tested for their ability to inhibit soluble BLyS (e.g., biotinylated BLyS)
from binding to
IM9 cells. In this assay, labeled soluble BLyS (e.g., biotinylated BLyS) is
incubated with
candidate anti-BLyS antibodies to allow for the formation of BLyS -anti-BLyS
antibody
complexes. Following incubation, an aliquot of the BLyS-anti-BLyS antibody
sample is
added to IM9 cells. The binding of soluble BLyS may be determined using
techniques
known in the art. For example, the binding of biotinylated BLyS to IM9 cells
may be
detected using a fluorimeter following the addition of streptavidin-delfia.
Biotinylated
BLyS, if it is not bound by antibodies that neutralize BLyS, binds to the
cells is detected.
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Thus, an antibody that decreases the amount of bio-BLyS that binds to IM-9
cells
(relative to a control sample in which the BLyS had been preincubated with an
irrelevant
antibody or no antibody at all) is identified as one that binds to and
neutralizes the soluble
form of BLyS. In another assay, antibodies are screened using ELISAs for those
antibodies that bind to biotinylated soluble BLyS, but do not bind membrane-
bound BLyS,
such as, for example, BLyS on membranes from U937 cells (see Examples 2 and 9,
ihfra).
In these assays, soluble BLyS (e.g., biotinylated BLyS) and membrane-bound
BLyS (e.g.,
on U937 membranes) are incubated in separate samples with the same antibodies
and
those antibodies that bind to the soluble BLyS (biotinylated BLyS), but not
membrane-
bound BLyS (e.g., on U937 membranes) are captured and identified.
[0341] Antibodies of the invention (including scFvs and other molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) may be
tested to
identify those antibodies that do not cross-react with APRIL, endokine-alpha,
VEGI,
TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS (see Example 4, ifzfra).
Antibodies may also be tested for their affinity for BLyS using, for example,
BIAcore
analysis (see Examples 6, 12, 17 and 18 infra). Antibodies may also be tested
for their
ability to stimulate, inhibit, or not alter, BLyS-induced immunoglobulin
production and/or
B-cell proliferation using techniques known to those of skill in the art. For
example,
human B-cells, BLyS and antibodies may be incubated together in 96 well plates
and 3H-
thymidine incorporation may be measured using a scintillation counter.
Selection and Screening for Antibodies that Immunospecifically Bind to
Membrane-bound
BLyS
[0342] Antibodies of the invention (including scFvs and other molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) may be
screened in a
variety of assays to identify those antibodies that immunospecifically bind to
the
membrane-bound form of BLyS. In one particular assay, antibodies that bind to
BLyS on
U937 membranes or immobilized histidine-tagged BLyS are captured. Other cell
lines that
express BLyS that might be useful for testing antibody binding to membrane-
bound form
of BLyS include, K-562, HL-60 and THP-1 cells. In another assay, antibodies
are
screened using ELISAs for those antibodies (or antibody fragments or variants)
that bind
to BLyS on U937 membranes or to histidine-tagged BLyS. In this assay,
antibodies are
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added to 96 well plates coated with U937 membranes or histidine-tagged BLyS
and those
antibodies or antibody fragments or variants that bind to the U937 membranes
or
histidine-tagged BLyS are captured. In another assay, antibodies are screened
using
ELISAs for those antibodies (or antibody fragments or variants thereof) that
do not bind to
biotinylated BLyS (soluble BLyS) but bind to membrane-bound BLyS, such as, for
example, that on membranes from U937 cells (see Example 2, infra). In these
assays,
soluble BLyS (e.g., biotinylated BLyS) and membrane-bound BLyS (e.g., on U937
membranes) are incubated in separate samples with the same antibodies (or
antibody
fragments or variants) and those antibodies (or antibody fragments or
variants) that do not
bind to the soluble BLyS (biotinylated BLyS), but bind the membrane-bound BLyS
(e.g.,
on U937 membranes) are captured and identified. In other assays, antibodies
are screened
using ELISAs to determine which of the antibodies (or antibody fragments or
variants)
that bind to histidine-tagged BLyS or membranes from U937 cells do not cross-
react with
APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS
(See Example 4, ii2fra). ELISAs can also be used to determine which of the
antibodies (or
antibody fragments or variants) that bind to histidine-tagged BLyS or
membranes. from
U937 cells bind to BLyS in the presence of TNF-alpha (see Example 4, infra).
Antibodies
or fragments or variants thereof that immunospecifically bind to the membrane-
bound
form of BLyS may also be tested for their affinity for histidine-tagged BLyS
using high-
throughput BIAcore analysis (see Example 14, infra).
[0343] Additionally, antibodies of the invention may be screened against cells
engineered to express an "uncleavable" form of BLyS in order to determine
their
specificity for the membrane-bound form of BLyS. Mutations in BLyS which may
achieve this result include, but are not limited to, the mutation or deletion
of amino acid
residues Lys-132 and/or Arg-133 of the BLyS sequence shown in SEQ ID N0:322~.
A
typical mutagenesis might include mutation of one or both of residues Lys-132
or Arg-133
to alanine residues. Cells expressing such an "uncleavable" form of BLyS
provide a
profound reagent to use in assaying the ability of antibodies to bind the
membrane-bound
form of BLyS.
Selection and Screening for Antibodies that Immunospecifically Bind to Soluble
and
Membrane-bound BLyS
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[0344] Antibodies of the invention (including scFvs and other molecules
comprising,
or alternately consisting of, antibody fragments or variants) may be screened
in a variety
of assays to identify those antibodies or antibody fragments or variants that
immunospecifically bind to the soluble form and membrane-bound form of BLyS.
In one
particular assay, antibodies that bind to immobilized BLyS are captured. In
another assay,
antibodies are screened using ELISAs for those antibodies (or antibody
fragments or
variants) that inhibit the binding of soluble BLyS (e.g. soluble bio-BLyS) to
IM-9 cells as
described supYa. In other assays, antibodies are screened using ELISAs for
those
antibodies that bind to membranes from U937 cells. Additionally, further ELISA
assays
may be performed using techniques known in the art to determine which
antibodies do not
cross-react with APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-
L,
LIGHT, and PBS, or those antibodies that bind to BLyS in the presence of TNF-
alpha (see
Example 4 i~zfra). Antibodies may be assayed in neutralization assays using
techniques
described herein or otherwise known in the art. Antibodies that
immunospecifically bind
to the soluble and membrane-bound forms of BLyS may also be tested for their
affinity for
BLyS using high-throughput BIAcore analysis.
Antibody Conjugates
[0345] The present invention encompasses antibodies (including scFvs and other
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), recombinantly fused or chemically conjugated (including both
covalent and non-
covalent conjugations) to a heterologous polypeptide (or portion thereof,
preferably at
least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at
least 70, at least 80, at
least 90 or at least 100 amino acids of the polypeptide) to generate fusion
proteins. The
fusion does not necessarily need to be direct, but may occur through linker
sequences. For
example, antibodies of the invention may be used to target heterologous
polypeptides to
particular cell types (e.g., cells of monocytic lineage and B-cells), either
in vitro or ifa vzvo,
by fusing or conjugating the heterologous polypeptides to antibodies of the
invention that
are specific for particular cell surface antigens (e.g., membrane-bound BLyS
on cells of
monocytic lineage) or which bind antigens that bind particular cell surface
receptors (e.g.,
TACI, BCMA, GAFF-R located on B cells). Antibodies fused or conjugated to
heterologous polypeptides may also be used in i~z vitro immunoassays and
purification
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methods using methods known in the art. See e.g., Harbor et al., supra, and
PCT
publication WO 93/2 1232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99
(1994);
U.S. Patent 5,474,981; Dillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol.
146:2446-2452 (1991), which are incorporated by reference in their entireties.
[0346] In one embodiment, a fusion protein comprises a polypeptide having an
amino
acid sequence of any one of the VH domains referred to in Table 1, and a
heterologous
polypeptide. In another embodiment, a fusion protein comprises a polypeptide
having the
amino acid sequence of any one of the VH CDRls referred to in Table 1, and a
heterologous polypeptide. In another embodiment, a fusion protein comprises a
polypeptide having the amino acid sequence of any one of the VH CDR2s referred
to in
Table l, and a heterologous polypeptide. In a preferred embodiment, a fusion
protein
comprises a polypeptide having the amino acid sequence of any one of the VH
CDR3s
referred to in Table 1 (i.e., SEQ ID NOS:2129 - 3227), and a heterologous
polypeptide.
[0347] In another embodiment, a fusion protein comprises a polypeptide having
the
amino acid sequence of any one of the VL domains referred to in Table 1, and a
heterologous polypeptide. In another embodiment, a fusion protein comprises a
polypeptide having the amino acid sequence of any one of the VL CDRls referred
to in
Table l, and a heterologous polypeptide. In yet another embodiment, a fusion,
protein
comprises a polypeptide having the amino acid sequence of any one of the VL
CDR2s
referred to in Table 1, and a heterologous polypeptide. In a preferred
embodiment, a
fusion protein comprises a polypeptide having the amino acid sequence of any
one of the
VL CDR3s referred to in Table 1, and a heterologous polypeptide.
[0348] In another embodiment, a fusion protein comprises a polypeptide having
the
amino acid sequence of any one of the VH domains referred to in Table l, and
one or
more VL domains referred to in Table 1, and a heterologous polypeptide. In
another
embodiment, a fusion protein of the present invention comprises a polypeptide
having the
amino acid sequence of any one of the VH CDRs referred to in Table l, and any
one of the
VL CDRs referred to in Table 1, and a heterologous polypeptide.
[0349] The present invention further includes compositions comprising, or
alternatively consisting of, heterologous polypeptides fused or conjugated to
antibody
fragments. For example, the heterologous polypeptides may be fused or
conjugated to a
Fab fragment, Fd fragment, Fv fragment, F(ab)Z fragment, or a portion thereof.
Methods
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for fusing or conjugating polypeptides to antibody portions are known in the
art. See, e.g.,
U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851;
5,112,946; EP
307,434; EP 367,166; PCT publications WO 96/04388; WO 9 1/06570; Ashkenazi et
al.,
Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol.
154:5590-
5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337- 11341 (1992)
(said
references incorporated by reference in their entireties).
[0350] Additional fusion proteins of the invention may be generated through
the
techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to
modulate the activities of antibodies (including scFvs and other molecules
comprising , or
alternatively consisting of, antibody fragments or variants thereof), such
methods can be
used to generate antibodies with altered activity (e.g., antibodies with
higher affinities and
lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793;
5,811,238;
5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion
Biotechnol. 8:724-
33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al.,
J. Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13
(1998)
(each of these patents and publications are hereby incorporated by reference
in its
entirety). In one embodiment, polynucleotides encoding antibodies of the
invention may
be altered by being subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In another
embodiment, one
or more portions of a polynucleotide encoding an antibody which portions
immunospecifically bind to BLyS may be recombined with one or more components,
motifs, sections, parts, domains, fragments, etc. of one or more heterologous
molecules.
[0351] Moreover, the antibodies of the present invention (including scFvs and
other
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), can be fused to marker sequences, such as a polypeptides to
facilitate
purification. In preferred embodiments, the marker amino acid sequence is a
hexa-
histidine polypeptide, such as the tag provided in a pQE vector (QIAGEN, Inc.,
9259 Eton
Avenue, Chatsworth, CA, 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824
(1989), for
instance, hexa-histidine provides for convenient purification of the fusion
protein. Other
peptide tags useful for purification include, but are not limited to, the
hemagglutinin "HA"
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tag, which corresponds to an epitope derived from the influenza hemagglutinin
protein
(Wilson et al., Cell 37:767 (1984)) and the "flag" tag (DYKDDDDK, (SEQ B7 No:
3238)
Stratagene, La Jolla, CA).
[0352] The present invention further encompasses antibodies (including scFvs
and
other molecules comprising, or alternatively consisting of, antibody fragments
or variants
thereof), conjugated to a diagnostic or therapeutic agent. The antibodies can
be used
diagnostically to, for example, monitor or prognose the development or
progression of a
tumor as part of a clinical testing procedure to, e.g., determine the efficacy
of a given
treatment regimen. Detection can be facilitated by coupling the antibody to a
detectable
substance. Examples of detectable substances include, but are not limited to,
various
enzymes, prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent
materials, radioactive materials, positron emitting metals using various
positron emission
tomographies, and nonradioactive paramagnetic metal ions. The detectable
substance may
be coupled or conjugated either directly to the antibody or indirectly,
through an
intermediate (such as, for example, a linker known in the art) using
techniques known in
the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can
be
conjugated to antibodies for use as diagnostics according to the present
invention.
Examples of ~ suitable enzymes include, but are not limited to, horseradish
peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of
suitable
prosthetic group complexes include, but are not limited to,
streptavidinlbiotin and
avidin/biotin; examples of suitable fluorescent materials include, but are not
limited to,
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material
includes, but is not limited to, luminol; examples of bioluminescent materials
include, but
are not limited to, luciferase, luciferin, and aequorin; and examples of
suitable radioactive
material include, but are not limited to, iodine (lslI' lash lash lalI)~
carbon (14C), sulfur
(35s)~ tritium (3H), indium (lls"'In, 113m~~ llaIn~ 111~)~ and technetium
(99Tc, ~9n'Tc),
thallium (ZOlTi), gallium (68Ga, 67Ga), palladium (losPd), molybdenum (~~Mo),
xenon
(ls3Xe), fluorine (18F), ls3Sm, 177Lu, 159Gd, 149Pm~ 14 175 166 90 47 18G
°La, Yb, Ho, Y, Sc, Re,
lssRe~ 142Pr~ 105Rh~ 97Ru~ 68Ge~ 57C~~ 65zn~ 85Sr~ 32P~ ls3Gd~ 169' slCr~
s4Mn~ 75Se~ 113Sn~
and 117Tin.
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[0353] Further, an antibody of the invention (including an scFv or other
molecule
comprising, or alternatively consisting of, antibody fragments or variants
thereof), may be
conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent,
a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as,
for example,
ai3Bi. In specific embodiments, antibodies of the invention are attached to
macrocyclic
chelators useful for conjugating radiometal ions, including but not limited
to, 111In, i77Lu,
9°Y, 166Iio, and lsssm, to polypeptides. In preferred embodiments, the
radiometal ion
associated with the macrocyclic chelators attached to antibodies of the
invention is lIn.
In preferred embodiments, the radiometal ion associated with the macrocyclic
chelators
attached to antibodies of the invention is 9°Y. In specific
embodiments, the macrocyclic
chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid
(DOTA). In other
specific embodiments, the DOTA is attached to the antibody of the invention
via a linker
molecule. Examples of linker molecules useful for conjugating DOTA to a
polypeptide
are commonly 'known in the art - see, for example, DeNardo et al., Clin Cancer
Res.
4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and
Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby
incorporated by
reference in their entirety.
[0354] A cytotoxin or cytotoxic agent includes any agent that is detrimental
to cells
and includes such molecules as small molecule toxins and enzymatically active
toxins of
bacterial, fungal, plant, or animal origin, or fragments thereof. Examples
include, but are
not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine,
mitomycin, etoposide (VP-16), tenoposide, vincristine, vinblastine, colchicin,
doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol,
thymidine kinase, endonuclease, RNAse, and puromycin and frragments, variants
or
homologs thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine),
alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine
(BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, ' and cisdichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics
(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and
anthramycin
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(AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine),
improsulfan,
piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine,
triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide
trimethylolomelamine, chlornaphazine, cholophosphamide, estramustine,
ifosfamide,
novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard,
chlorozotocin,
fotemustine, nimustine, ranimustine, aclacinomysins, azaserine, cactinomycin,
calichearnicin, carabicin, carminomycin, carzinophilin, chromomycins,
detorubicin,
6-diazo-5-oxo-L-norleucine, epirubicin, esorubicin, idarubicin, marcellomycin,
mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
quelamycin,
rodorubicin, streptonigrin, tubercidin, ubenimex, zinostatin, zorubicin,
denopterin,
pteropterin, trimetrexate, fludarabine, thiamiprine, ancitabine, azacitidine,
6-azauridine,
carmofur, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU,
calusterone,
dromostanolone propionate, epitiostanol, mepitiostane, testolactone,
aminoglutethimide,
mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside,
aminolevulinic
acid, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine,
dernecolcine, diaziquone,
elfornithine, elliptiniurn acetate, etoglucid, gallium nitrate, hydroxyurea,
lentinan,
lonidamine, mitoguazone, mopidamol, nitracrine, pentostatin, phenamet,
pirarubicin,
podophyllinic acid, 2-ethylhydrazide, procarbazine, PSI~O, razoxane,
sizofiran,
spirogermanium, tenuazonic acid, triaziquone, 2, 2',2"-trichlorotriethylamine,
urethan,
vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,
gacytosine,
arabinoside ("Ara-C"), taxoids, e.g. paclitaxel (TAXOL", Bristol-Myers Squibb
Oncology,
Princeton, NJ) doxetaxel (TAXOTERE", Rh6ne-Poulenc Rorer, Antony, France),
gemcitabine, ifosfamide, vinorelbine, navelbine, novantrone, teniposide,
aminopterin,
xeloda, ibandronate, CPT-I 1, topoisomerase inhibitor RFS 2000,
difluoromethylornithine
(DMFO), retinoic acid, esperamicins, capecitabine, and pharmaceutically
acceptable salts,
acids or derivatives of any of the above. Also included in this definition are
anti-hormonal
agents that act to regulate or inhibit hormone action on tumors such as anti-
estrogens
including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-
imidazoles, 4
hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, toremifene
(Fareston),
and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,
and goserelin,
and pharmaceutically acceptable salts, acids or derivatives of any of the
above.
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[0355] Techniques known in the art may be applied to label antibodies of the
invention. Such techniques include, but are not limited to, the use of
bifunctional
conjugating agents (see e.g., U.S. Patent Nos. 5,756,065; 5,714,631;
5,696,239; 5,652,361;
5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560;
and
5,808,003; the contents of each of which are hereby incorporated by reference
in its
entirety) and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-T
reaction).
[0356] The antibodies of the invention which are conjugates can be used for
modifying a given biological response, the therapeutic agent or drug moiety is
not to be
construed as limited to classical chemical therapeutic agents. For example,
the drug
moiety may be a protein or polypeptide possessing a desired biological
activity. Such
proteins may include, but are not limited to, for example, a toxin such as
abrin, ricin A,
alpha toxin, pseudomonas exotoxin, or diphtheria toxin, saporin, momordin,
gelonin,
pokeweed antiviral protein, alpha-sarcin and cholera toxin; a protein such as
tumor
necrosis factor, alpha-interferon, beta-interferon, nerve growth factor,
platelet derived
growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-
alpha, TNF-
beta, AIM I (see, International Publication No. WO 97133899), AIM II (see,
International
Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol.,
6:1567-1574
(1994)), VEGI (see, International Publication No. WO 99/23105), a thrombotic
agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological
response modifiers
such as, for example, lymphokines, interleukin-1 (IL- 1), interleukin-2 (IL-
2), interleukin-
6 (1L-6), granulocyte macrophage colony stimulating factor (GM-CSF),
granulocyte
colony stimulating factor (G-CSF), or other growth factors.
[0357] Antibodies of the invention (including scFvs and other molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof), may
also be
attached to solid supports, which are particularly useful for immunoassays or
purification
of the target antigen. Such solid supports include, but are not limited to,
glass, cellulose,
polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
[0358] Techniques for conjugating a therapeutic moiety to antibodies are well
known,
see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs
In Cancer
Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-
56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug
Delivery", in
Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel
Dekker,
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Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:
A
Review", in Monoclonal Antibodies '84: Biological And Clinical Applications,
Pinchera
et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective
Of The
Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16
(Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic
Properties Of
Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).
[0359] Alternatively, an antibody of the invention can be conjugated to a
second
antibody to form an antibody heteroconjugate as described by Segal in U.S.
Patent No.
4,676,980, which is incorporated herein by reference in its entirety.
[0360] An antibody of the invention (including an scFv or and other molecule
comprising, or alternatively consisting of, an antibody fragment or variant
thereof), with
or without a therapeutic moiety conjugated to it, administered alone or in
combination
with cytotoxic factors) and/or cytokine(s) can be used as a therapeutic.
Use of Antibodies for Epitope Mapping
[0361] The present invention provides antibodies (including scFvs and other
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof), that can be used to identify epitopes of BLyS. In particular, the
antibodies of the
present invention can be used to identify epitopes of human BLyS (SEQ ID
NOS:3228
and/or 3229) or BLyS expressed on human monocytes; murine BLyS (SEQ ID
NOS:3230
and/or 3231) or BLyS expressed on murine monocytes; rat BLyS (either the
soluble forms
as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane
associated
form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the monkey
BLyS
polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey BLyS,
or
BLyS expressed on monkey monocytes)using techniques described herein or
otherwise
known in the art. Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-
5135
(1985), further described in U.S. Patent No. 4,631,211.)
Diagnostic Uses of Antibodies
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[0362] Labeled antibodies of the invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) which
specifically bind
to BLyS can be used for diagnostic purposes to detect, diagnose, prognose, or
monitor
diseases and/or disorders associated with the aberrant expression and/or
activity of BLyS
or BLyS receptor. The invention provides for the detection of aberrant
expression of BLyS
comprising: (a) assaying the expression of BLyS in a biological sample from an
individual using one or more antibodies of the invention that
immunospecifically binds to
BLyS; and (b) comparing the level of BLyS with a standard level of BLyS, e.g.,
in normal
biological samples, whereby an increase or decrease in the assayed level of
BLyS
compared to the standard level of BLyS is indicative of aberrant expression.
[0363] By "biological sample" is intended any fluids and/or cells obtained
from an
individual, body fluid, body tissue, body cell, cell line, tissue culture, or
other source
which may contain BLyS protein or mRNA. Body fluids include, but are not
limited to,
sera, plasma, urine, synovial fluid, spinal fluid, saliva, and mucous. Tissues
samples may
be taken from virtually any tissue in the body. Tissue samples may also be
obtained from
autopsy material. Methods for obtaining tissue biopsies and body fluids from
mammals
are well known in the art. Where the biological sample is to include mRNA, a
tissue
biopsy is the preferred source.
[0364] The invention also provides for the detection of aberrant expression of
BLyS
receptor comprising (a) assaying the expression of BLyS receptor in a
biological sample
from an individual using one or more antibodies or fragments or variants
thereof that
immunospecifically binds only to soluble BLyS, but does not inhibit BLyS BLyS
receptor
binding. Such an antibody, by way of an example that is not to be construed as
limiting,
would be one that is able to capture a biotinylated BLyS from solution (see
Example 8),
but that would not prevent BLyS from binding to IM-9 cells (see Example 3).
and (b)
comparing the level of BLyS receptor with a standard level of BLyS receptor,
e.g., in
normal tissue or cell samples, whereby an increase or decrease in the assayed
level of
BLyS receptor compared to the standard level of BLyS receptor is indicative of
aberrant
expression.
[0365] Antibodies of the invention (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof) which specifically bind
to BLyS can
be used for diagnostic purposes to detect, diagnose, prognose, or monitor
autoimmune
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disorders and/or immunodeficiencies, and/or diseases or conditions associated
therewith.
The invention provides for the detection of aberrant expression of BLyS
comprising: (a)
assaying the expression of BLyS in a biological sample from an individual
using one or
more antibodies of the invention that imrriunospecifically binds to BLyS; and
(b)
comparing the level of BLyS with a standard level of BLyS, e.g., in normal
biological
samples, whereby an increase or decrease in the assayed level of BLyS compared
to the
standard level of BLyS is indicative of an autoimmune disorder or disease
and/or an
immunodeficiency. In specific embodiments, an increase in the assayed level of
BLyS is
indicative of an autoimmune disorder or disease. In other specific
embodiments, a
decrease in the assayed level of BLyS is indicative of an immunodeficiency.
[0366] Antibodies of the invention (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof) which specifically bind
to BLyS but,
do not inhibit BLyS/BLyS receptor binding can be used for diagnostic purposes
to detect,
diagnose, prognose, or monitor autoimmune disorders and/or immunodeficiencies,
and/or
diseases or conditions associated therewith. The invention provides for the
detection of
aberrant expression of BLyS receptor comprising: (a) assaying the expression
of BLyS
receptor in a biological sample from an individual using one or more
antibodies of the
invention that immunospecifically binds to BLyS; and (b) comparing the level
of BLyS
receptor with a standard level of BLyS receptor, e.g., in normal biological
samples,
whereby an increase or decrease in the assayed level of BLyS receptor compared
to the
standard level of BLyS receptor is indicative of an autoimmune disorder or
disease and/or
an immunodeficiency. In specific embodiments, an increase in the assayed level
of BLyS
receptor is indicative of an autoimmune disorder or disease. In other specific
embodiments, a decrease in the assayed level of BLyS receptor is indicative of
an
immunodeficiency.
[0367] Autoimmune disorders, diseases, or conditions that may be detected,
diagnosed, prognosed, or monitored using the antibodies of the invention
include, but are
not limited to, autoimmune hemolytic anemia, autoimmune neonatal
thrombocytopenia,
idiopathic thrombocytopenia purpura, autoimmune neutropenia,
autoimmunocytopenia,
hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive
enteropathy,
allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic
heart disease,
glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis,
Uveitis
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Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's
Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis,
IgA
glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-
Barre
Syndrome, diabetes mellitus (e.g. Type I diabetes mellitus or insulin
dependent diabetes
mellitis), juvenile onset diabetes, and autoimmune inflammatory eye,
autoimmune
thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus
erhythematosus,
discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such
as, for
example, (a) Graves' Disease , (b) Myasthenia Gravis, and (c) insulin
resistance,
autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid
arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue
disease,
polymyositis/dermatomyositis, pernicious anemia (Addison's disease),
idiopathic
Addison's disease, infertility, glomerulonephritis such as primary
glomerulonephritis and
IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes millitus,
and
adrenergic drug resistance (including adrenergic drug resistance with asthma
or cystic
fibrosis), chronic active hepatitis, primary biliary cirrhosis, other
endocrine gland failure,
vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic
dermatitis, asthma,
inflammatory myopathies, and other inflammatory, granulomatous, degenerative,
and
atrophic disorders and other disorders such as inflammatory skin diseases
including
psoriasis 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), meningitis, encephalitis, colitis,
allergic conditions
such as eczema and other conditions involving infiltration of T cells and
chronic
inflammatory responses, atherosclerosis, leukocyte adhesion deficiency,
Reynaud's
syndrome, and immune responses associated with acute and delayed
hypersensitivity
mediated by cytokines and T-lymphocytes typically found in tuberculosis,
sarcoidosis,
granulomatosis and diseases involving leukocyte diapedesis, central nervous
system
(CNS) inflammatory disorder, multiple organ injury syndrome, antigen-antibody
complex
mediated diseases, anti-glomerular basement membrane disease, Lambert-Eaton
myasthenic syndrome, Beheet disease, giant cell arteritis, immune complex
nephritis, IgA
nephropathy, IgM polyneuropathies or autoimmune thrombocytopenia etc.
[0368] In specific embodiments, the present invention encompasses methods and
compositions for detecting, diagnosing and/or prognosing diseases or disorders
associated
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with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some
immunodeficiencies). In other specific embodiments, the present invention
encompasses
methods and compositions for detecting, diagnosing and/or prognosing diseases
or
disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency).
[0369] Immunodeficiencies that may be detected, diagnosed, prognosed, or
monitored
using the antibodies of the invention include, but are not limited to, severe
combined
immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase
deficiency
(ADA deficiency), X-linked agammaglobulinemia (XLA), Breton's disease,
congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia,
. dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia
of
infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common
variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked
immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM,
selective IgA deficiency, IgG subclass deficiency (with or without IgA
deficiency),
antibody deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig
heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative
disorder
(BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss
type),
reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia,
thymic
alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-
telangiectasia, short
limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-
combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency
(PNP),
MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined
immunodeficiency.
[0370] Elevated levels of soluble BLyS have been observed in the serum of
patients
with Systemic Lupus Erythematosus (SLE). In comparing the sera of 150 SLE
patients
with that of 38 control individuals, it was found that most of the SLE
patients had more
than 5ng/ml of serum BLyS, more than 30% of SLE patients had levels greater
than
lOng/ml, and approximately 10% of SLE patients had serum BLyS levels greater
than
20ng/ml. In contrast, the majority of normal controls had BLyS levels less
than 5ng/ml,
and less than 10% had levels higher than lOng/ml. The elevated levels of BLyS
protein in
sera is present in the soluble form and has biologic activity as assayed by
the ability to
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stimulate anti-IgM treated B cells in vitro. SLE patients with more than
l5nglml serum
BLyS were also found to have elevated levels of anti-dsDNA antibodies compared
to both
normal controls and SLE patients with less than 5ng/ml of serum
BLyS.(unpublished
data).
[0371] In addition the serum of two subgroups of patients which were positive
for
anti-nuclear antibodies (ANA+) but did not meet the formal requirements of the
American
College of Rheumatology (ACR) for classification of SLE were analyzed for BLyS
levels.
The first subgroup of sera was ANA+ sera that came from patients who did not
present
with the clinical impression of SLE. This group had only slightly elevated
levels of BLyS
(~9ng/ml BLyS). The second subgroup however, which was ANA+ sera from patients
who presented with the clinical impression of SLE, had significantly increased
BLyS
levels (~l5ng/ml). These results suggest that an elevated level of BLyS
precedes the
formal fulfillment of the ACR criteria. The ACR criteria are described in Tan,
E.M., et al,
Arthritis and Rheumatism 25:1271-1277 (1982).
v
[0372] Thus in specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor Systemic Lupus Erythematosus or conditions associated therewith. The
invention
provides for the detection of aberrant expression of BLyS comprising: (a)
assaying the
expression of BLyS in a biological sample of an individual using one or more
antibodies
of the invention that immunospecifically binds to BLyS; and (b) comparing the
level of
BLyS with a standard level of BLyS, e.g., in normal biological samples,
whereby an
increase in the assayed level of BLyS compared to the standard level of BLyS
is indicative
of SLE.
[0373] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor IgA nephropathy or conditions associated therewith. The invention
provides for
the detection of aberrant expression of BLyS comprising: (a) assaying the
expression of
BLyS in a biological sample of an individual using one or more antibodies of
the
invention that immunospecifically binds to BLyS; and (b) comparing the level
of BLyS
with a standard level of BLyS, e.g., in normal biological samples, whereby an
increase in
the assayed level of BLyS compared to the standard level of BLyS is indicative
of IgA
nephropathy.
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[0374] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor Sjogren's Syndrome or conditions associated therewith. The invention
provides
for the detection of aberrant expression of BLyS comprising: (a) assaying the
expression
of BLyS in a biological sample of an individual using one or more antibodies
of the
invention that immunospecifically binds to BLyS; and (b) comparing the level
of BLyS
with a standard level of BLyS, e.g., in normal biological samples, whereby an
increase in
the assayed level of BLyS compared to the standard level of BLyS is indicative
of
Sjogren's Syndrome.
[0375] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor HIV infection or conditions associated therewith (e.g. AIDS). The
invention
provides for the detection of aberrant expression of BLyS comprising: (a)
assaying the
expression of BLyS in a biological sample of an individual using one or more
antibodies
of the invention that immunospecifically binds to BLyS; and (b) comparing the
level of
BLyS with a standard level of BLyS, e.g., in normal biological samples,
whereby an
increase in the assayed level of BLyS compared to the standard level of BLyS
is indicative
of HIV infection.
[0376] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor Myasthenia Gravis or conditions associated therewith. The invention
provides for
the detection of aberrant expression of BLyS comprising: (a) assaying the
expression of
BLyS in a biological sample of an individual using one or more antibodies of
the
invention that immunospecifically binds to BLyS; and (b) comparing the level
of BLyS
with a standard level of BLyS, e.g., in normal biological samples, whereby an
increase in
the assayed level of BLyS compared to the standard level of BLyS is indicative
of
Myasthenia Gravis.
[0377] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor idiopathic thrombocytopenic purpura (ITP) or conditions associated
therewith.
The invention provides for the detection of aberrant expression of BLyS
comprising: (a)
assaying the expression of BLyS in a biological sample of an individual using
one or more
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antibodies of the invention that immunospecifically binds to BLyS; and (b)
comparing the
level of BLyS with a standard level of BLyS, e.g., in normal biological
samples, whereby
an increase in the assayed level of BLyS compared to the standard level of
BLyS is
indicative of idiopathic thrombocytopenic purpura (ITP).
[0378] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor hemolytic anemia or conditions associated therewith. The invention
provides for
the detection of aberrant expression of BLyS comprising: (a) assaying the
expression of
BLyS in a biological sample of an individual using one or more antibodies of
the
invention that immunospecifically binds to BLyS; and (b) comparing the level
of BLyS
with a standard level of BLyS, e.g., in normal biological samples, whereby an
increase in
the assayed level of BLyS compared to the standard level of BLyS is indicative
of
hemolytic anemia.
[0379] In other specific embodiments, antibodies' of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor thyroiditis or conditions associated therewith. The invention provides
for the
detection of aberrant expression of BLyS comprising: (a) assaying the
expression of BLyS
in a biological sample of an individual using one or more antibodies of the
invention that
immunospecifically binds to BLyS; and (b) comparing the level of BLyS with a
standard
level of BLyS, e.g., in normal biological samples, whereby an increase in the
assayed level
of BLyS compared to the standard level of BLyS is indicative of thyroiditis.
[0380] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect, diagnose,
prognose, or
monitor Goodpasture's syndrome or conditions associated therewith. The
invention
provides for the detection of aberrant expression of BLyS comprising: (a)
assaying the
expression of BLyS in a biological sample of an individual using one or more
antibodies
of the invention that immunospecifically binds to BLyS; and (b) comparing the
level of
BLyS with a standard level of BLyS, e.g., in normal biological samples,
whereby an
increase in the assayed level of BLyS compared to the standard level of BLyS
is indicative
of Goodpasture's syndrome.
[0381] In other specific embodiments, antibodies of the invention which
specifically
bind to BLyS can be used for diagnostic purposes to detect; diagnose,
prognose, or
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monitor multiple sclerosis or conditions associated therewith. The invention
provides for
the detection of aberrant expression of BLyS comprising: (a) assaying the
expression of
BLyS in a biological sample of an individual using one or more antibodies of
the
invention that immunospecifically binds to BLyS; and (b) comparing the level
of BLyS
with a standard level of BLyS, e.g., in normal biological samples, whereby an
increase in
the assayed level of BLyS compared to the standard level of BLyS is indicative
of
multiple sclerosis.
[0382] In additional embodiments, antibodies of the invention which
specifically bind
to BLyS can be used for diagnostic purposes to detect, diagnose, prognose, or
monitor
Rheumatoid Arthritis. The invention provides for the detection of aberrant
expression of
BLyS comprising: (a) assaying the expression of BLyS in a biological sample
(e.g., serum
and synovial fluid) of an individual using one or more antibodies of the
invention that
immunospecifically binds to BLyS; and (b) comparing the level of BLyS with a
standard
level of BLyS, e.g., in normal biological samples, whereby an increase in the
assayed level
of BLyS compared to the standard level of BLyS is indicative of Rheumatoid
arthritis.
[0383] In additional embodiments, antibodies of the invention which
specifically bind
to BLyS can be used for diagnostic purposes to detect, diagnose, prognose, or
monitor an
immune-based rheumatologic disease, (e.g., SLE, rheumatoid arthritis, CREST
syndrome
(a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon,
esophageal
motility disorders, sclerodactyly, and telangiectasia.), Seronegative
spondyloarthropathy
(SpA), Polymyositis/dermatomyositis, Microscopic polyangiitis, Hepatitis C-
associated
arthritis, Takayasu's arteritis, and undifferentiated connective tissue
disorder). The
invention provides for the detection of aberrant expression of BLyS
comprising: (a)
assaying the expression of BLyS in a biological sample (e.g., serum and
synovial fluid) of
an individual using one or more antibodies of the invention that
immunospecifically binds
to BLyS; and (b) comparing the level of BLyS with a standard level of BLyS,
e.g., in
normal biological samples, whereby an increase in the assayed level of BLyS
compared to
the standard level of BLyS is indicative of monitor an immune-based
rheumatologic
disease.
[0384] It has been observed, that serum BLyS levels inversely correlate with
nephrotic
range proteinuria (>3gm proteinuria in a 24 hour urine collection) using a
sample of 71
SLE patients (p=0.019). Proteinuria was determined in 71 SLE patients within
one month
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of phlebotomy for serum BLyS determination. Serum BLyS was classified as low,
normal, or high based on the 5~' through 95~' percentiles for normal controls.
Nephrotic-
range proteinuria was inversely correlated with serum Neutrokine-alpha levels.
Thus, in
specific embodiments, serum levels of BLyS (determined using one or more
antibodies of
the present invention) in individuals diagnosed with an immune based
rheumatologic
disease (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of
scleroderma
characterized by calcinosis, Raynaud's phenomenon, esophageal motility
disorders,
sclerodactyly, and telangiectasia.), seronegative spondyloarthropathy (SpA),
polymyositis/dermatomyositis, microscopic polyangiitis, hepatitis C-asociated
arthritis,
Takayasu's arteritis, and undifferentiated connective tissue disorder) may be
used to
determine, diagnose, prognose, or monitor the severity of certain aspects or
symptoms of
the disease, such as nephrotic-range proteinuria.
[0385] In another specific embodiment, antibodies of the invention are used to
diagnose, prognose, treat, or prevent conditions associated with CVID,
including, but not
limited to, conditions associated with acute and recurring infections (e.g.,
pneumonia,
bronchitis, sinusitis, otitis media, sepsis, meningitis, septic arthritis, and
osteomyelitis),
chronic lung disease, autoimmunity, granulomatous disease, lymphoma, cancers
(e.g.,
cancers of the breast, stomach, colon, mouth, prostate, lung, vagina, ovary,
skin, and
melanin forming cells (i.e. melanoma), inflammatory bowel disease (e.g.,
Crohn's disease,
ulcerative colitis, and ulcerative proctitis), malabsorption, Hodgkin's
disease, and
Waldenstrom's macroglobulinemia.
[0386] The invention provides a diagnostic assay for diagnosing or prognosing
a
disease or disorder, comprising: (a) assaying for the level of BLyS in a
biological sample
of an individual using one or more antibodies of the invention that
immunospecifically
bind to BLyS; and (b) comparing the level of BLyS with a standard BLyS level,
e.g., in a
biological sample from a patient without the disease or disorder, whereby an
increase or
decrease in the assayed BLyS level compared to the standard level of BLyS is
indicative
of a particular disease or disorder. With respect to cancer, the presence of a
relatively high
amount of BLyS in biopsied tissue from an individual may indicate a
predisposition for
the development of the disease, or may provide a means for detecting the
disease prior to
the appearance of actual clinical symptoms. A more definitive diagnosis of
this type may
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allow health professionals to employ preventative measures or aggressive
treatment earlier
thereby preventing the development or further progression of the cancer.
[0387] In specific embodiments, the presence of a relatively high amount of
membrane-bound BLyS in a biological sample is indicative of monocytic cell
related
leukemias or lymphomas, such as, for example acute myelogenous leukemia and/or
the
severity thereof.
[0388] In other specific embodiments, the presence of a relatively high amount
of
BLyS receptor in a biological sample (as determined using antibodies of the
invention that
bind to soluble BLyS, but do not inhibit BLyS/BLyS receptor binding) is
indicative of B
cell related leukemias or lymphomas (e.g., chronic lymphocytic leukemia,
multiple
myeloma, non-Hodgkin's lymphoma, and Hodgkin's disease), and/or the severity
thereof.
[0389] In specific embodiments, the invention provides a diagnostic assay for
diagnosing or prognosing Systemic Lupus Erythematosus, comprising: (a)
assaying for the
level of BLyS in a biological sample of an individual using one or more
antibodies of the
invention that immunospecifically bind to BLyS; and (b) comparing the level of
BLyS
with a standard BLyS level, e.g., in a biological sample from a patient
without Systemic
Lupus Erythematosus, whereby an increase in the assayed BLyS level compared to
the
standard level of BLyS is indicative of Systemic Lupus Erythematosus.
[0390] In specific embodiments, the invention provides a diagnostic assay for
diagnosing or prognosing a Rheumatoid Arthritis, comprising: (a) assaying for
the level of
BLyS in a biological sample of an individual using one or more antibodies of
the
invention that immunospecifically bind to BLyS; and (b) comparing the level of
BLyS
with a standard BLyS level, e.g., in a biological sample from a patient
without
Rheumatoid Arthritis, whereby an increase or decrease in the assayed BLyS
level
compared to the standard level of BLyS is indicative of Rheumatoid Arthritis.
[0391] The invention provides a diagnostic assay for diagnosing or prognosing
a
disease or disorder, comprising: (a) assaying for the level of BLyS receptor
in cells or a
tissue sample of an individual using one or more antibodies of the invention
that
immunospecifically binds only to soluble BLyS, but does not neutralize BLyS
/BLyS
receptor binding; and (b) comparing the level of BLyS receptor with a standard
BLyS
receptor level, e.g., in a tissue sample from a patient without the disease or
disorder,
whereby an increase or decrease in the assayed BLyS receptor level compared to
the
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standard level of BLyS receptor is indicative of a particular disease or
disorder. With
respect to cancer, the presence of a relatively high amount of BLyS receptor
in biopsied
tissue from an individual may indicate a predisposition for the development of
the disease,
or may provide a means for detecting the disease prior to the appearance of
actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to
employ preventative measures or aggressive treatment earlier thereby
preventing the
development or further progression of the cancer.
[0392] Antibodies of the invention (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof) can be used to assay
protein levels in
a biological sample using classical immunohistological methods as described
herein or as
known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.
101:976-985
(1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096 (1987)). Other
antibody-based
methods useful for detecting protein gene expression include immunoassays,
such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable
antibody assay labels are known in the art and include enzyme labels, such as,
glucose
oxidase, alkaline phosphatase, and horseradish peroxidase; radioisotopes, such
as iodine
(i2ih 123h l2sh 131~~ c~.bon (14C), sulfur (3sS), tritium (3I~, indium (111In,
112In, u3~In,
usmIn), technetiumlJ (9~TC,~9'T'Tc), thallium (Z°1Ti), gallium (68Ga,
67Ga), palladium (lo3Pd),
molybdenum (~9Mo), xenon (133Xe), fluorine (18F), ls3Sm, 177Lu, ls~Gd, 149Pm,
i4oLa,
i7s~~ 166H~~ ~o~,~ 47Sc, 186Re, 1$$Re, 142Pr, 1°sRh, and 97Ru;
luminescent labels, such as
luminol; and fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0393] One aspect of the invention is the detection and diagnosis of a disease
or
disorder associated with aberrant expression of BLyS or BLyS receptor in an
animal,
preferably a mammal and most preferably a human. In one embodiment, diagnosis
comprises: a) administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled antibody of
the invention
(including molecules comprising, or alternatively consisting of, antibody
fragments or
variants thereof) that immunospecifically binds to BLyS; b) waiting for a time
interval
following the administering for permitting the labeled antibody to
preferentially
concentrate at sites in the subject where BLyS is expressed (and for unbound
labeled
molecule to be cleared to background level); c) determining background level;
and d)
detecting the labeled antibody in the subject, such that detection of labeled
antibody or
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fragment thereof above the background level and above or below the level
observed in a
person without the disease or disorder indicates that the subject has a
particular disease or
disorder associated with aberrant expression of BLyS or BLyS receptor.
Background
level can be determined by various methods including, comparing the amount of
labeled
molecule detected to a standard value previously determined for a particular
system.
[0394] It will be understood in the art that the size of the subject and the
imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic
images. In the case of a radioisotope moiety, for a human subject, the
quantity of
radioactivity injected will normally range from about 5 to 20 millicuries of
99Tc. The
labeled antibody will then preferentially accumulate at the location of cells
which contain
the specific protein. In vivo tumor imaging is described in S.W. Burchiel et
al.,
"Imxnunopharmacokinetics of Radiolabeled Antibodies and Their Fragments."
(Chapter
13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and
B. A.
Rhodes, eds., Masson Publishing Inc. (1982).
[0395] Depending on several variables, including the type of label used and
the mode
of administration, the time interval following the administration for
permitting the labeled
molecule to preferentially concentrate at sites in the subject and for unbound
labeled
molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours
or 6 to 12
hours. In another embodiment the time interval following administration is 5
to 20 days or
to 10 days.
[0396] In an embodiment, monitoring of the disease or disorder is carried out
by
repeating the method for diagnosing the disease or disorder, for example, one
month after
initial diagnosis, six months after initial diagnosis, one year after initial
diagnosis, etc.
[0397] Presence of the labeled molecule can be detected in the patient using
methods
known in the art for i~c vivo scanning. These methods depend upon the type of
label used.
Skilled artisans will be able to determine the appropriate method for
detecting a particular
label. Methods and devices that may be used in the diagnostic methods of the
invention
include, but are not limited to, computed tomography (CT), whole body scan
such as
position emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0398] In a specific embodiment, the molecule is labeled with a radioisotope
and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al.,
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U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled
with a
fluorescent compound and is detected in the patient using a fluorescence
responsive
scanning instrument. In another embodiment, the molecule is labeled with a
positron
emitting metal and is detected in the patient using positron emission-
tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic label and is
detected in
a patient using magnetic resonance imaging (MRI).
Immunophenotypin~
[0399] The antibodies of the invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) may be
utilized for
immunophenotyping of cell lines and biological samples by their BLyS
expression or
BLyS receptor expression. Various techniques can be utilized using antibodies,
fragments, or variants of the invention to screen for cellular populations
(i.e., immune
cells, particularly monocytic cells or B-cells) expressing BLyS or BLyS
receptor, and
include magnetic separation using antibody-coated magnetic beads, "panning"
with
antibody attached to a solid matrix (i.e., plate), and flow cytometry (see,
e.g., U.S. Patent
5,95,660; and Morrison et al., Cell, 96:737-49 (1999)).
[0400] These techniques allow for the screening of particular populations of
cells,
such as might be found with hematological malignancies (i.e., minimal residual
disease
(MRD) in acute leukemic patients) and "non-self' cells in transplantations to
prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for
the
screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation
and/or differentiation, as might be found in human umbilical cord blood.
[0401] In one embodiment, antibodies of the invention (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) are used
to identify cells of monocytic or B cell origin.
Ther~eutic Uses of Antibodies
[0402] The present invention is further directed to antibody-based therapies
which
involve administering antibodies of the invention (including molecules
comprising, or
alternatively consisting of, antibody fragments or variants thereof) to an
animal, preferably
a mammal, and most preferably a human; patient for treating one or more of the
disclosed
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diseases, disorders, or conditions. Therapeutic compounds of the invention
include, but
are not limited to, antibodies of the invention and nucleic acids encoding
antibodies (and
anti-idiotypic antibodies) of the invention as described herein. The
antibodies of the
invention can be used to treat, ameliorate or prevent diseases, disorders or
conditions
associated with aberrant expression and/or activity of BLyS or BLyS receptor,
including,
but not limited to, any one or more of the diseases, disorders, or conditions
described
herein. The treatment and/or prevention of diseases, disorders, or conditions
associated
with aberrant BLyS expression and/or activity or aberrant BLyS receptor
expression
and/or activity includes, but is not limited to, alleviating symptoms
associated with those
diseases, disorders or conditions. Antibodies of the invention may be provided
in
pharmaceutically acceptable compositions as known in the art or as described
herein.
[0403] Antibodies of the present invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) that
function as
agonists or antagonists of BLyS, preferably of BLyS-induced signal
transduction, can be
administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant BLyS expression, lack of BLyS function, aberrant BLyS receptor
expression, or lack of BLyS receptor function. For example, antibodies of the
invention
which disrupt the interaction between BLyS and its receptor may be
administered to an
animal to treat, prevent or ameliorate a disease or disorder associated with
aberrant BLyS
expression, excessive BLyS function, aberrant BLyS receptor expression, or
excessive of
BLyS receptor function. Antibodies of the invention which do not prevent BLyS
from
binding its receptor but inhibit or downregulate BLyS-induced signal
transduction can be
administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant BLyS expression, excessive BLyS function, aberrant BLyS receptor
expression, or excessive BLyS receptor function. In particular, antibodies of
the present
invention which prevent BLyS-induced signal transduction by specifically
recognizing the
unbound BLyS, receptor-bound BLyS or both unbound and receptor-bound BLyS can
be
administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant BLyS expression, excessive BLyS function, aberrant BLyS receptor
expression, or excessive BLyS receptor function. The ability of an antibody of
the
invention to inhibit or downregulate BLyS-induced signal transduction may be
determined
by techniques described herein or otherwise known in the art. For example,
BLyS-
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induced receptor activation and the activation of signaling molecules can be
determined by
detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the
receptor or a
signaling molecule by immunoprecipitation followed by western blot analysis
(for
example, as described herein).
[0404] In a specific embodiment, an antibody of the present invention
(including
molecules comprising, or alternatively consisting of, antibody fragments or
variants
thereof) that inhibits or downregulates BLyS activity by at least 95%, at
least 90%, at least
85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at
least 45%, at
least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least
20%, or at least
10% relative to BLyS activity in absence of the antibody is administered to an
animal to
treat, prevent or ameliorate a disease or disorder associated with aberrant
BLyS
expression, excessive BLyS function, aberrant BLyS receptor expression, or
excessive
BLyS receptor function. In another embodiment, a combination of antibodies, a
combination of antibody fragments, a combination of antibody variants, or a
combination
of antibodies, antibody fragments, and/or variants that inhibit or
downregulate BLyS
activity by at least 95%, at least 90%, at least 85%, at least 80%, at least
75%, at least
70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at
least 40%, at
least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least
10% relative to
BLyS activity in absence of said antibodies, antibody fragments, and/or
antibody variants
are administered to an animal to treat, prevent or ameliorate a disease or
disorder
associated with aberrant BLyS expression, excessive BLyS function, aberrant
BLyS
receptor expression, or excessive BLyS receptor function.
[0405] Further, ,antibodies of the present invention (including molecules
comprising,
or alternatively consisting of, antibody fragments or variants thereof) which
activate
BLyS-induced signal transduction can be administered to an animal to treat,
prevent or
ameliorate a disease or disorder associated with aberrant BLyS expression,
lack of BLyS
function, aberrant BLyS receptor expression, or lack of BLyS receptor
function. These
antibodies may potentiate or activate either all or a subset of the biological
activities of
BLyS-mediated receptor activation, for example, by inducing multimerization of
BLyS
and/or multimerization of the receptor. The antibodies of the invention may be
administered with or without being pre-complexed with BLyS. In a specific
embodiment,
an antibody of the present invention that increases BLyS activity by at least
5%, at least
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10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least
75°70, at least 80%, at least 85%, at least 90%, at least 95%, or at
least 99% relative to
BLyS activity in absence of the antibody is administered to an animal to
treat, prevent or
ameliorate a disease or disorder associated with aberrant BLyS expression,
lack of BLyS
function, aberrant BLyS receptor expression, or lack of BLyS receptor
function. In
another embodiment, a combination of antibodies, a combination of antibody
fragments, a
combination of antibody variants, or a combination of antibodies, antibody
fragments
and/or antibody variants that increase BLyS activity by at least 5%, at least
10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least
80%, at least 85%, at least 90%, at least 95%, or at least 99% relative to
BLyS activity in
absence of the said antibodies or antibody fragments and/or antibody variants
is
administered to an animal to treat, prevent or ameliorate a disease or
disorder associated
with aberrant BLyS expression or lack of BLyS function or aberrant BLyS
receptor
expression or lack of BLyS receptor function.
[0406] ~ne or more antibodies of the present invention (including molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) that
immunospecifically bind to BLyS may be used locally or systemically in the
body as a
therapeutic. The antibodies of this invention (including molecules comprising,
or
alternatively consisting of, antibody fragments or variants thereof) may also
be
advantageously utilized in combination with other monoclonal or chimeric
antibodies, or
with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3
and IL-7), for
example, which serve to increase the number or activity of effector cells
which interact
with the antibodies.
[0407] The antibodies of the invention (including molecules comprising, or
alternatively consisting of, antibody fragments or variants thereof) may be
administered
alone or in combination with other types of treatments (e.g., radiation
therapy,
chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents, anti-
angiogenesis
and anti-inflammatory agents). Generally, administration of products of a
species origin
or species reactivity (in the case of antibodies) that is the same species as
that of the
patient is preferred. Thus, in a preferred embodiment, human antibodies,
fragments, or
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variants, (e.g., derivatives), or nucleic acids, are administered to a human
patient for
therapy or prophylaxis.
[0408] It is preferred to use high affinity and/or potent in vivo inhibiting
and/or
neutralizing antibodies of the invention (including molecules comprising, or
alternatively
consisting of, antibody fragments or variants thereof) that immunospecifically
bind to
BLyS, or polynucleotides encoding antibodies that immunospecifically bind to
BLyS, for
both immunoassays directed to and therapy of disorders related to BLyS
polynucleotides
or polypeptides, including fragments thereof. Such antibodies will preferably
have an
affinity for BLyS and/or BLyS fragments. Preferred binding affinities include
those with
a dissociation constant or KD less than or equal to 5 X 10-2 M, 10-2 M, 5 X 10-
3 M,10-3 M, 5
X 10-4 M, 10-4 M, 5 X 10-5 M, or 10-5 M. More preferably, antibodies of the
invention
bind BLyS polypeptides or fragments or variants thereof with a dissociation
constant or
KD less than or equal to 5 X 10-6 M, 10-6 M, 5 X 10-7 M, 10-7 M, 5 X 10-$ M,
or 10-8 M.
Even more preferably, antibodies of the invention bind BLyS polypeptides or
fragments or
variants thereof with a dissociation constant or KD less than or equal to 5 X
10-~ M,10-9 M,
X 10-i° M, 10-1° M, 5 X 10-11 M, 10-11 M, 5 X 10-is M, 10-12 M,
5 X -is M,10-13 M, 5 X
10-14 M, 10-14 M, 5 X 10-1j M, or 10-15 M. The invention encompasses
antibodies that bind
BLyS polypeptides with a dissociation constant or KD that is within any one of
the ranges
that are between each of the individual recited values.
[0409] In a preferred embodiment, antibodies of the invention neutralize BLyS
activity. In another preferred embodiment, antibodies of the invention inhibit
B cell
proliferation.
[0410] In a preferred embodiment, antibodies of the invention (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) inhibit
or reduce binding of the soluble form of BLyS to a BLyS receptor. In another
preferred
embodiment antibodies of the invention inhibit or reduce B cell proliferation
induced by
the soluble form of BLyS. In another preferred embodiment antibodies of the
invention
inhibit or reduce immunoglobulin production induced by the soluble form of
BLyS.
[0411] In a preferred embodiment" antibodies of the invention (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) inhibit
or reduce binding of membrane-bound BLyS to a BLyS receptor. In another
preferred
embodiment, antibodies of the invention inhibit or reduce B cell proliferation
induced by
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the membrane-bound form of BLyS. In another preferred embodiment, antibodies
of the
invention inhibit or reduce immunoglobulin production induced by the membrane
bound
form of BLyS.
[0412] In a preferred embodiment, antibodies of the invention (including
molecules
comprising, or alternatively consisting of, antibody fragments or variants
thereof) inhibit
or reduce binding of both the soluble and membrane-bound forms of BLyS to a
BLyS
receptor. In another preferred embodiment, antibodies of the invention inhibit
or reduce B
cell proliferation induced by either or both forms of BLyS. In another
preferred
embodiment, antibodies of the invention inhibit or reduce immunoglobulin
production
induced by either or both forms of BLyS.
[0413] ~ In one embodiment, the invention provides a method of delivering
antibody
conjugates of the invention to targeted cells, such as, for example, monocytic
cells
expressing the membrane-bound form of BLyS, or B cells expressing a BLyS
receptor.
[0414] In one embodiment, the invention provides a method for the specific
delivery
of antibodies and antibody conjugates of the invention to cells by
administering molecules
of the invention that are associated with heterologous polypeptides or nucleic
acids. In
one example, the invention provides a method for delivering a therapeutic
protein into the
targeted cell. In another example, the invention provides a method for
delivering a single
stranded nucleic acid (e.g., antisense or ribozymes) or double stranded
nucleic acid (e.g.,
DNA that can integrate into the cell's genome or replicate episomally and that
can be
transcribed) into the targeted cell.
[0415] In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by administering
antibodies or
antibody conjugates of the invention (e.g., antibodies conjugated with
radioisotopes,
toxins, or cytotoxic prodrugs).In a specific embodiment, the invention
provides a method
for the specific destruction of cells of monocytic lineage (e.g., monocytic
cell related
leukemias or lymphomas, such as, for example acute myelogenous leukemia) by
administering antibodies or antibody conjugates of the invention (e.g.,
antibodies
conjugated with radioisotopes, toxins, or cytotoxic prodrugs) that
immunospecifically bind
the membrane-bound form of BLyS. In another specific embodiment, the invention
provides a method for the specific destruction of cells of B cell lineage
(e.g., B cell related
leukemias or lymphomas (e.g., chronic lymphocytic leukemia, multiple myeloma,
non-
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Hodgkin's lymphoma, and Hodgkin's disease) by administering antibodies or
antibody
conjugates of the invention (e.g., antibodies conjugated with radioisotopes,
toxins, or
cytotoxic prodrugs) that bind soluble BLyS, but do not inhibit BLyS binding to
a BLyS
receptor on B cells.
[0416] In another preferred embodiment antibodies of the invention (including
antibody fragments and variants) promote or enhance B cell proliferation
induced by the
soluble form of BLyS. In another preferred embodiment, antibodies of the
invention
(including antibody fragments and variants) promote or enhance B cell
proliferation
induced by the membrane or soluble form of APRIL. In another preferred
embodiment
antibodies of the invention (including antibody fragments and variants)
increase or
enhance immunoglobulin production induced by the soluble form of BLyS. In
another
preferred embodiment antibodies of the invention (including antibody fragments
and
variants) increase or enhance immunoglobulin production induced by the
membrane
bound or soluble form of APRIL. In another preferred embodiment antibodies of
the
invention (including antibody fragments and variants) increase or enhance
immunoglobulin production in response to T cell dependent immunogens. In
another
preferred embodiment antibodies of the invention (including antibody fragments
and
variants, and anti-antibody antibodies) increase or enhance immunoglobulin
production in
response to T cell independent immunogens.
[0417] In another embodiment, therapeutic or pharmaceutical compositions of
the
invention are administered to an animal to treat, prevent or ameliorate immune
disorders.
Immune disorders include, but are not limited to, autoimmune disorders (e.g.,
arthritis,
graft rejection, Hashimoto's thyroiditis, insulin-dependent diabetes, lupus,
idiopathic
thrombocytopenic purpura, systemic lupus erythrematosus and multiple
sclerosis), elective
IgA deficiency, ataxia-telangiectasia, common variable immunodeficiency
(CVID), X-
linked agammaglobulinemia, severe combined immunodeficiency (SCID), Wiskott-
Aldrich syndrome, idiopathic hyper-eosinophilic syndrome, monocytic leukemoid
reaction, monocytic leukocytosis, monocytic leukopenia, monocytopenia,
monocytosis,
and graft or transplant rejection.
[0418] As discussed herein, antibodies and antibody compositions of the
invention,
may be used to treat, prevent, ameliorate, diagnose or prognose various immune
system-related disorders and/or conditions associated with these disorders, in
mammals,
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preferably humans. Many autoimmune disorders result from inappropriate
recognition of
self as foreign material by immune cells. This inappropriate recognition
results in an
immune response leading to the destruction of the host tissue. Therefore, the
administration of antibody and antibody compositions of the invention that can
inhibit an
immune response, particularly the proliferation of B cells and/or the
production of
immunoglobulins, may be an effective therapy in treating and/or preventing
autoimmune
disorders. Thus, in preferred embodiments, antibodies and antibody
compositions of the
invention are used to treat, prevent, ameliorate, diagnose and/or prognose an
autoimmune
disorder, or conditions) associated with such disorder.
[0419] Autoimmune disorders and conditions associated with these disorders
that may
be treated, prevented, ameliorated, diagnosed and/or prognosed with the
therapeutic and
pharmaceutical compositions of the invention include, but are not limited to,
autoimmune
hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia
purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic
encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart
disease, '
glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis,
Uveitis
Ophthalmic, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's
Disease, Stiff Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis,
IgA
glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-
Barre
Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye
disease.
[0420] Additional autoimmune disorders and conditions associated with these
disorders that may be treated, prevented, ameliorated, diagnosed and/or
prognosed with
the therapeutic and pharmaceutical compositions of the invention include, but
are not
limited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis) (often
characterized, e.g., by cell-mediated and humoral thyroid cytotoxicity),
systemic lupus
erhythematosus (often characterized, e.g., by circulating and locally
generated immune
complexes), discoid lupus; Goodpasture's syndrome (often characterized, e.g.,
by anti-
basement membrane antibodies), Pemphigus (often characterized, e.g., by
epidermal
acantholytic antibodies), Receptor autoimmunities such as, for example, (a)
Graves'
Disease (often characterized, e.g., by TSH receptor antibodies), (b)
Myasthenia Gravis
(often characterized, e.g., by acetylcholine receptor antibodies), and (c)
insulin resistance
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(often characterized, e.g., by insulin receptor antibodies), autoimmune
hemolytic anemia
(often characterized, e.g., by phagocytosis of antibody-sensitized RBCs),
autoimmune
thrombocytopenic purpura (often characterized, e.g., by phagocytosis of
antibody-
sensitized platelets.
[0421] Additional autoimmune disorders and conditions associated with these
disorders that may be treated, prevented, ameliorated, diagnosed and/or
prognosed with
the therapeutic and pharmaceutical compositions of the invention include, but
are not
limited to, rheumatoid arthritis (often characterized, e.g., by immune
complexes in joints),
schleroderma with anti-collagen antibodies (often characterized, e.g., by
nucleolar and
other nuclear antibodies), mixed connective tissue disease (often
characterized, e.g., by
antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)),
polymyositis/dermatomyositis (often characterized, e.g., by nonhistone ANA),
pernicious
anemia (often characterized, e.g., by antiparietal cell, microsomes, and
intrinsic factor
antibodies), idiopathic Addison's disease (often characterized, e.g., by
humoral and cell-
mediated adrenal cytotoxicity, infertility (often characterized, e.g., by
antispermatozoal
antibodies), glomerulonephritis (often characterized, e.g., by glomerular
basement
membrane antibodies or immune complexes) such as primary glomerulonephritis
and IgA
nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and
complement in
basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple
tissue
antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus (often
characterized, e.g., by cell-mediated and humoral islet cell antibodies), and
adrenergic
drug resistance (including adrenergic drug resistance with asthma or cystic
fibrosis) (often
characterized, e.g., by beta-adrenergic receptor antibodies), chronic active
hepatitis (often
characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis
(often
characterized, e.g., by mitchondrial antibodies), other endocrine gland
failure (often
characterized, e.g., by specific tissue antibodies in some cases), vitiligo
(often
characterized, e.g., by melanocyte antibodies), vasculitis (often
characterized, e.g., by Ig
and complement in vessel walls and/or low serum complement), post-MI (often
characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often
characterized,
e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG
and IgM
antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and
IgM antibodies
to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE),
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inflammatory myopathies, and many other inflammatory, granulomatous,
degenerative,
and atrophic disorders.
[0422] In a preferred embodiment, therapeutic and pharmaceutical compositions
of the
invention, are used to treat, prevent, ameliorate, diagnose or prognose, a
member of the
group: autoimmune hemolytic anemia, as primary glomerulonephritis, IgA
glomerulonephritis, Goodpasture's syndrome, idiopathic thrombocytopenia,
Multiple
Sclerosis, Myasthenia Gravis, Pemphigus, polymyositis/dermatomyositis,
relapsing
polychondritis, rheumatoid arthritis, Sjogren's syndrome, systemic lupus
erythematosus,
Uveitis, vasculitis,and primary biliary cirrhosis.
[0423] In another preferred embodiment, therapeutic and pharmaceutical
compositions
of the invention, are used to treat, prevent, ameliorate, diagnose or
prognose, an immune
based-rheumatologic disease, such as, for example, SLE, rheumatoid arthritis,
CREST
syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's
phenomenon,
esophageal motility disorders, sclerodactyly, and telangiectasia.),
Seronegative
spondyloarthropathy (SpA), polymyositis/ dermatomyositis, microscopic
polyangiitis,
hepatitis C-associated arthritis, Takayasu's arteritis, and undifferentiated
connective tissue
disorder.
[0424] In a specific preferred embodiment, therapeutic and pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
rheumatoid arthritis and/or medical conditions associated therewith.
[0425] For example, an antibody, or antibodies, of the present invention are
used to
treat patients with clinical diagnosis of rheumatoid arthritis (RA). The
patient treated
preferably will not have a B cell malignancy. Moreover, the patient is
optionally further
treated with any one or more agents employed for treating RA such as
salicylate;
nonsteroidal anti-inflammatory drugs such as indomethacin, phenylbutazone,
phenylacetic
acid derivatives (e.g. ibuprofen and fenoprofen), naphthalene acetic acids
(naproxen),
pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated
anthranilic acid
(meclofenamate sodium), piroxicam, zomepirac and diflunisal; antimalarials
such as
chloroquine; gold salts; penicillamine; or immunosuppressive agents such as
methotrexate
or corticosteroids in dosages known for such drugs or reduced dosages.
Preferably
however, the patient is only treated with an antibody, or antibodies, of the
present
invention. Antibodies of the present invention are administered to the RA
patient
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according to a dosing schedule as described infra, which may be readily
determined by
one of ordinary skill in the art. The primary response is determined by the
Paulus index
(Paulus et al. Athritis Rheum. 33:477-484 (1990)), i.e. improvement in morning
stiffness,
number of painful and inflamed joints, erythrocyte sedimentation (ESR), and at
least a
2-point improvement on a 5-point scale of disease severity assessed by patient
and by
physician. Administration of an antibody, or antibodies, of the present
invention will
alleviate one or more of the symptoms of RA in the patient treated as
described above.
[0426] In a specific preferred embodiment, therapeutic and pharmaceutical
compositions of the invention, are used to treat, prevent, amelioate, diagnose
or prognose,
lupus andlor medical conditions associated therewith. Lupus-associated
conditions that
may be treated, prevented, ameliorated, prognosed and/or diagnosed with the
antibodies
and antibody compositions of the invention include, but are not limited to,
hematologic
disorders (e.g., hemolytic anemia, leukopenia, lymphopenia, and
thrombocytopenia),
immunologic disorders (e.g., anti-DNA antibodies, and anti-Sm antibodies),
rashes,
photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss,
serositis (e.g.; pleuritus
(pleurisy)), renal disorders (e.g., nephritis), neurological disorders (e.g.,
seizures,
peripheral neuropathy, CNS related disorders), gastroinstestinal disorders,
Raynaud
phenomenon, and pericarditis. In a preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention are used to treat, prevent, ameliorate,
diagnose, or prognose,
renal disorders associated with systemic lupus erythematosus. In a most
preferred
embodiment, therapeutic and pharmaceutical compositions of the invention are
used to
treat, prevent, ameliorate, diagnose, or prognose, nephritis associated with
systemic lupus
erythematosus. In another most preferred embodiment, therapeutic or
pharmaceutical
compositions of the invention are administered to an animal to treat, prevent
or ameliorate
lupus or glomerular nephritis.
[0427] In a further specific embodiment, antibodies of the invention are used
to treat,
inhibit, prognose, diagnose or prevent hemolytic anemia. For example, patients
diagnosed
with autoimmune hemolytic anemia (AIHA), e.g., cryoglobinernia or Coombs
positive
anemia, are treated with an antibody, or antibodies, of the present invention.
AIHA is an
acquired hemolytic anemia due to auto-antibodies that react with the patient's
red blood
cells. The patient treated preferably will not have a B cell malignancy.
Further adjunct
therapies (such as glucocorticoids, prednisone, azathioprine,
cyclophosphamide,
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vinca-laden platelets or Danazol) may be combined with the antibody therapy,
but
preferably the patient is treated with an antibody, or antibodies, of the
present invention as
a single-agent throughout the course of therapy. Antibodies of the present
invention are
administered to the hemolytic anemia patient according to a dosing schedule as
described
infra, which may be readily determined by one of ordinary skill in the art.
Overall
response rate is determined based upon an improvement in blood counts,
decreased
requirement for transfusions, improved hemoglobin levels and/or a decrease in
the
evidence of hemolysis as determined by standard chemical parameters.
Administration of
an antibody, or antibodies of the present invention will improve any one or
more of the
symptoms of hemolytic anemia in the patient treated as described above. For
example, the
patient treated as described above will show an increase in hemoglobin and an
improvement in chemical parameters of hemolysis or return to normal as
measured by
serum lactic dehydrogenase and/or bilirubin.
[0428] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
Sjogren's Syndrome and/or medical conditions associated therewith.
[0429] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
HIV infection and/or medical conditions associated therewith (e.g. AIDS).
[0430] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
Myasthenia gravis and/or medical conditions associated therewith.
[0431] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
IgA nephropathy and/or medical conditions associated therewith.
[0432] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
hemolytic anemia and/or medical conditions associated therewith.
[0433] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
thyroiditis and/or medical conditions associated therewith.
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[0434] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
Goodpasture's Syndrome and/or medical conditions associated therewith.
[0435] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
multiple sclerosis and/or medical conditions associated therewith.
[0436] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
chronic lymphocytic leukemia (CLL) and/or medical conditions associated
therewith.
[0437] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
multiple myeloma and/or medical conditions associated therewith.
[0438] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
Non-Hodgkin's lymphoma and/or medical conditions associated therewith.
[0439] In another specific preferred embodiment, therapeutic and
pharmaceutical
compositions of the invention, are used to treat, prevent, ameliorate,
diagnose or prognose,
Hodgkin's disease and/or medical conditions associated therewith.
[0440] In another specific embodiment, antibodies of the invention are used to
treat,
inhibit, prognose, diagnose or prevent adult immune thrombocytopenic purpura.
Adult
immune thrombocytopenic purpura (ITP) is a relatively rare hematologic
disorder that
constitutes the most common of the immune-mediated cytopenias. The disease
typically
presents with severe thrombocytopenia that may be associated with acute
hemorrhage in
the presence of normal to increased megakaryocytes in the bone marrow. Most
patients
with ITP have an IgG antibody directed against target antigens on the outer
surface of the
platelet membrane, resulting in platelet sequestration in the spleen and
accelerated
reticuloendothelial destruction of platelets (Bussell, J.B. Hematol. Oncol.
Clin. North Am.
(4):179 (1990)). A number of therapeutic interventions have been shown to be
effective in
the treatment of ITP. Steroids are generally considered first-line therapy,
after which most
patients are candidates for intravenous immunoglobulin (IVIG), splenectomy, or
other
medical therapies including vincristine or immunosuppressive/cytotoxic agents.
Up to
80% of patients with ITP initially respond to a course of steroids, but far
fewer have
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complete and lasting remissions. Splenectomy has been recommended as standard
second-line therapy for steroid failures, and leads to prolonged remission in
nearly 60% of
cases yet may result in reduced immunity to infection. Splenectomy is a major
surgical
procedure that may be associated with substantial morbidity (15%) and
mortality (2%).
IVIG has also been used as second line medical therapy, although only a small
proportion
of adult patients with ITP achieve remission. Therapeutic options that would
interfere
with the production of autoantibodies by activated B cells without the
associated
morbidities that occur with corticosteroids and/or splenectomy would provide
an
important treatment approach for a proportion of patients with TTP. Patients
with clinical
diagnosis of ITP are treated with an antibody, or antibodies of the present
invention,
optionally in combination with steroid therapy. The patient treated will not
have a B cell
malignancy. Antibodies of the present invention are administered to the RA
patient
according to a dosing schedule as described infra, which may be readily
determined by
one of ordinary skill in the art. Overall patient response rate is determined
based upon a
platelet count determined on two consecutive occasions two weeks apart
following
treatments as described above. See, George et al. "Idiopathic Thrombocytopenic
Purpura:
A Practice Guideline Developed by Explicit Methods for The American Society of
Hematology", Blood 88:3-40 (1996), expressly incorporated herein by reference.
[0441] In another embodiment, therapeutic or pharmaceutical compositions of
the
invention are administered to an animal to treat, prevent or ameliorate an IgE-
mediated
allergic reaction or histamine-mediated allergic reaction. Examples of
allergic reactions
include, but are not limited to, asthma, rhinitis, eczema, chronic urticaria,
and atopic
dermatitis. In another embodiment, therapeutic or pharmaceutical compositions
of the
invention are administered to an animal to treat, prevent, or ameliorate
anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group incompatibility. In
another
embodiment, therapeutic or pharmaceutical compositions of the invention are
administered to an animal to treat, prevent or ameliorate or modulate
inflammation or an
inflammatory disorder. Examples of chronic and acute inflammatory disorders
that may
be treated prevented or ameliorated with the therapeutic and pharmaceutical
compositions
of the invention include, but are not limited to, chronic prostatitis,
granulomatous
prostatitis and malacoplakia, inflammation associated with infection (e.g.,
septic shock,
sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-
reperfusion injury,
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endotoxin lethality, arthritis, complement-mediated hyperacute rejection,
nephritis,
cytokine or chemokine induced lung injury, Crohn's disease, inflammatory bowel
disease,
chronic and acute inflammatory pulmonary diseases, bacterial infection,
psoriasis,
septicemia, cerebral malaria, arthritis, gastroenteritis, and glomerular
nephritis.
[0442] In another embodiment, therapeutic or pharmaceutical compositions of
the
invention are administered to an animal to treat, prevent or ameliorate
ischemia and
arteriosclerosis. Examples of such disorders include, but are not limited to,
reperfusion
damage (e.g., in the heart and/or brain) and cardiac hypertrophy.
[0443] Therapeutic or pharmaceutical compositions of the invention, may also
be
administered to modulate blood clotting and to treat or prevent blood clotting
disorders,
such as, for example, antibody-mediated thrombosis (i.e., antiphospholipid
antibody
syndrome (APS)). For example, therapeutic or pharmaceutical compositions of
the
invention, may inhibit the proliferation and differentiation of cells involved
in producing
anticardiolipin antibodies. These compositions of the invention can be used to
treat,
prevent, ameliorate, diagnose, and/or prognose thrombotic related events
including, but
not limited to, stroke (and recurrent stroke), heart attack, deep vein
thrombosis, pulmonary
embolism, myocardial infarction, coronary artery disease (e.g., antibody -
mediated
coronary artery disease), thrombosis, graft reocclusion following
cardiovascular surgery
(e.g., coronary arterial bypass grafts, recurrent fetal loss, and recurrent
cardiovascular
thromboembolic events.
[0444] Therapeutic or pharmaceutical compositions of the invention, may also
be
administered to treat, prevent, or ameliorate organ rejection or graft-versus-
host disease
(GVHI~) and/or conditions associated therewith. Organ rejection occurs by host
immune
cell destruction of the transplanted tissue through an immune response.
Similarly, an
immune response is also involved in GVHD, but, in this case, the foreign
transplanted
immune cells destroy the host tissues. The administration of antibodies of the
invention,
that inhibit an immune response, may be an effective therapy in preventing
organ rejection
or GVHD.
[0445] In another embodiment, therapeutic or pharmaceutical compositions of
the
invention are administered to an animal to treat, prevent or ameliorate a
disease or
disorder diseases associated with increased apoptosis including, but not
limited to, AIDS,
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease,
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Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration),
myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as
that caused
by myocardial infarction, stroke and reperfusion injury), toxin-induced liver
disease (such
as that caused by alcohol), septic shock, cachexia and anorexia. In another
embodiment,
therapeutic or pharmaceutical compositions of the invention are administered
to an animal
to treat, prevent or ameliorate bone marrow failure, for example, aplastic
anemia and
myelodysplastic syndrome.
[0446] In another embodiment, therapeutic or pharmaceutical compositions of
the
invention are administered to an animal to treat, prevent or ameliorate
growth,
progression, and/or metastases of malignancies and proliferative disorders
associated with
increased cell survival, or the inhibition of apoptosis. Examples of such
disorders,
include, but are not limited to, leukemia (e.g., acute leukemia such as acute
lymphocytic
leukemia and acute myelocytic leukemia), neoplasms, tumors (e.g.,
fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon
carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous
cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,
cervical
cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder
carcinoma,
epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, melanoma, neuroblastoma, and retinoblastoma), heavy chain disease,
metastases, or any disease or disorder characterized by uncontrolled cell
growth.
[0447] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used to treat or prevent a disorder characterized by
hpergammagloulinemia
(e.g., A>DS, autoimmune diseases, and some immunodeficiencies).
[0448] In a specific embodiment, therapeutic or pharmaceutical compositions of
the '
invention are used to treat or prevent a disorder characterized by deficient
serum
immunoglobulin production, recurrent infections, and/or immune system
dysfunction.
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Moreover, therapeutic or pharmaceutical compositions of the invention may be
used to
treat or prevent infections of the joints, bones, skin, and/or parotid glands,
blood-borne
infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis),
autoimmune
diseases (e.g., those disclosed herein), inflammatory disorders, and
malignancies, and/or
any disease or disorder or condition associated with these infections,
diseases, disorders
and/or malignancies) including, but not limited to, CVID, other primary immune
deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media,
conjunctivitis,
pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster),
and/or
pneumocystis carnii.
[0449] Therapeutic or pharmaceutical compositions of the invention of the
invention
thereof, may be used to diagnose, prognose, treat or prevent one or more of
the following
diseases or disorders, or conditions associated therewith: primary
immuodeficiencies,
immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant
(e.g.,
recent bone marrow transplant in adults or children), chronic B-cell
lymphocytic
leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic
inflammatory
demyelinating polyneuropathy, and post-transfusion purpura.
[0450] Additionally, therapeutic or pharmaceutical compositions of the
invention may
be used to diagnose, progriose, treat or prevent one or more of the following
diseases,
disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia
(e.g.,
anemia associated with parvovirus B 19, patients with stable multiple myeloma
who are at
high risk for infection (e.g., recurrent infection), autoimmune hemolytic
anemia (e.g.,
warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal
thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g.,
cytomegalovirus (CMV)-negative recipients of CMV-positive organs),
hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with risk factor
for
infection or morbidity), epilepsy (e.g., intractable epilepsy), systemic
vasculitic
syndromes, myasthenia gravis (e.g., decompensation in myasthenia gravis),
dermatomyositis, and polymyositis.
[0451] Additional preferred embodiments of the invention include, but are not
limited
to, the use of therapeutic or pharmaceutical compositions of the invention in
the following
applications:
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[0452] Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea
pig, pigs,
micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human
primate, and
human, most preferably human) to boost the immune system to produce increased
quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher
affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to
increase an immune
response. In a specific nonexclusive embodiment, therapeutic or pharmaceutical
compositions of the invention are administered to boost the immune system to
produce
increased quantities of IgG. In another specific nonexclusive embodiment,
antibodies of
the are administered to boost the immune system to produce increased
quantities of IgA.
In another specific nonexclusive embodiment antibodies of the invention are
administered
to boost the immune system to produce increased quantities of IgM.
[0453] Administration to an animal (including, but not limited to, those
listed above,
and also including transgenic animals) incapable of producing functional
endogenous
antibody molecules or having an otherwise compromised endogenous immune
system, but
which is capable of producing human immunoglobulin molecules by means of a
reconstituted or partially reconstituted immune system from another animal
(see, e.g.,
published PCT Application Nos. W098/24893, WO/9634096, WO/9633735, and
WO/9110741 ).
[0454] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a vaccine adjuvant that enhances immune responsiveness
to specific
antigen. In a specific embodiment, the vaccine is an antibody described
herein. In another
specific embodiment, the vaccine adjuvant is a polynucleotide described herein
(e.g., an
antibody polynucleotide genetic vaccine adjuvant). As discussed herein,
therapeutic or
pharmaceutical compositions of the invention may be administered using
techniques
known in the art, including but not limited to, liposomal delivery,
recombinant vector
delivery, injection of naked DNA, and gene gun delivery.
[0455] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
V
invention are used as an adjuvant to enhance tumor-specific immune responses.
[0456] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an adjuvant to enhance anti-viral immune responses. Anti-
viral
immune responses that may be enhanced using the compositions of the invention
as an
adjuvant, include, but are not limited to, virus and virus associated diseases
or symptoms
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described herein or otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an immune
response to a
virus, disease, or symptom selected from the group consisting of: AIDS,
meningitis,
Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific
embodiment, the
compositions of the invention are used as an adjuvant to enhance an immune
response to a
virus, disease, or symptom selected from the group consisting of: HIV/AIDS,
Respiratory
syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and
B,
Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift
Valley fever,
Herpes simplex, and yellow fever. In another specific embodiment, the
compositions of
the invention are used as an adjuvant to enhance an immune response to the HIV
gp120
antigen.
[0457] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an adjuvant to enhance anti-bacterial or anti-fungal
immune
responses. Anti-bacterial or anti-fungal immune responses that may be enhanced
using the
compositions of the invention as an adjuvant, include bacteria or fungus and
bacteria or
fungus associated diseases or symptoms described herein or otherwise known in
the art.
In specific embodiments, the compositions of the invention are used as an
adjuvant to
enhance an immune response to a bacteria or fungus, disease, or symptom
selected from
the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
In another
specific embodiment, the compositions of the invention are used as an adjuvant
to enhance
an immune response to a bacteria or fungus, disease, or symptom selected from
the group
consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,
Salmonella
paratyphi, Neisseria meningitidis, Streptococcus pneumoniae, Group B
streptococcus,
Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli,
Borrelia
burgdorferi, and Plasmodium (malaria).
[0458] In, a specific embodiment, therapeutic or pharmaceutical compositions
of the
invention are used as an adjuvant to enhance anti-parasitic immune responses.
Anti-
parasitic immune responses that may be enhanced using the compositions of the
invention
as an adjuvant, include parasite and parasite associated diseases or symptoms
described
herein or otherwise known in the art. In specific embodiments, the
compositions of the
invention are used as an adjuvant to enhance an immune response to a parasite.
In another
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specific embodiment, the compositions of the invention are used as an adjuvant
to enhance
an immune response to Plasmodium (malaria).
[0459] In a specific embodiment, compositions of the invention may be
administered
to patients as vaccine adjuvants. In a further specific embodiment,
compositions of the
invention may be administered as vaccine adjuvants to patients suffering from
an immune-
deficiency. In a further specific embodiment, compositions of the invention
may be
administered as vaccine adjuvants to patients suffering from HIV.
[0460] In a specific embodiment, compositions of the invention may be used to
increase or enhance antigen-specific antibody responses to standard and
experimental
vaccines. In a specific embodiment, compositions of the invention may be used
to enhance
seroconversion in patients treated with standard and experimental vaccines. In
another
specific embodiment, compositions of the invention may be used to increase the
repertoire
of antibodies recognizing unique epitopes in response to standard and
experimental
vaccination.
[0461] In a preferred embodiment, antibodies of the invention (including
antibody
fragments and variants, and anti-antibody antibodies) increase or enhance
antigen-specific
antibody responses to standard and experimental vaccines by regulating binding
of the
soluble form of BLyS to a BLyS receptor (e.g., TACI - GenBank accession number
AAC51790; BCMA - GenBank accession number I~-001183; and/or BAFF-R -
GenBank accession number NP 443177). In another preferred embodiment,
antibodies of
the invention (including antibody fragments and variants, and anti-antibody
antibodies)
increase or enhance antigen-specific antibody responses to standard and
experimental
vaccines by regulating binding of the soluble form of APRIL to an APRIL
receptor (e.g.,
BCMA and TACI).
[0462] In a preferred embodiment, antibodies of the invention (including
antibody
fragments and variants, and anti-antibody antibodies) increase or enhance
seroconversion
in patients treated with standard and experimental vaccines by regulating
binding of the
soluble form of BLyS to BLyS receptor (e.g., TACI - .GenBank accession number
AAC51790; BCMA - GenBank accession number NP 001183; and/or BAFF-R -
GenBank accession number NP 443177). In another preferred embodiment,
antibodies of
the invention (including antibody fragments and variants, and anti-antibody
antibodies)
increase or enhance seroconversion in patients treated with standard and
experimental
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vaccines by regulating binding of the soluble form of APRIL to an APRIL
receptor (e.g.,
BCMA and TACI).
[0463] In a preferred embodiment, antibodies of the invention (including
antibody
fragments and variants, and anti-antibody antibodies) increase or enhance the
repertoire of
antibodies recognizing unique epitopes in response to standard and
experimental
vaccination by regulating binding of the soluble form of BLyS to a BLyS
receptor (e.g.,
TACI - GenBank accession number AAC51790; BCMA - GenBank accession number
~ 001183; and/or BAFF-R - GenBank accession number NP 443177). In another
preferred embodiment, antibodies of the invention (including antibody
fragments and
variants, and anti-antibody antibodies) increase or enhance the repertoire of
antibodies
recognizing unique epitopes in response to standard and experimental
vaccination by
regulating binding of the soluble form of APRIL to an APRIL receptor (e.g.,
BCMA and
TACI).
[0464] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a stimulator of B cell responsiveness to pathogens.
[0465] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent that elevates the immune status of an
individual prior to
their receipt of immunosuppressive therapies.
[0466] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent to induce higher affinity antibodies.
[0467] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent to increase serum immunoglobulin
concentrations.
[0468] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent to accelerate recovery of immunocompromised
individuals.
[0469] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent to boost immunoresponsiveness among aged
populations.
[0470] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an immune system enhancer prior to, during, or after
bone marrow
transplant and/or other transplants (e.g., allogeneic or xenogeneic organ
transplantation).
,With respect to transplantation, compositions of the invention may be
administered prior
to, concomitant with, and/or after transplantation. In a specific embodiment,
compositions
of the invention are administered after transplantation, prior to the
beginning of recovery
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of T-cell populations. In another specific embodiment, compositions of the
invention are
first administered after transplantation after the beginning of recovery of T
cell
populations, but prior to full recovery of B cell populations.
[0471] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent to boost immunoresponsiveness among B cell
immunodeficient individuals, such as, for example, an individual who has
undergone a
partial or complete splenectomy. B cell immunodeficiencies that may be
ameliorated or
treated by administering the antibodies and/or compositions of the invention
include, but
are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-
autosomal,
adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia
(XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile
agammaglobulinemia, acquired agammaglobulinemia, adult onset
agammaglobulinemia,
late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia,
agammaglobulinemia, common variable immunodeficiency (CVID) (acquired),
Wiskott-
Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass
deficiency
(with or without IgA deficiency), antibody deficiency with normal or elevated
Igs,
immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain
deficiency, B
cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency,
recessive
agammaglobulinernia (Swiss type), reticular dysgenesis, neonatal neutropenia,
severe
congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency,
ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative
syndrome
(XLP), Nezelof syndrome-combined imrnunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte
Syndrome)
and severe combined immunodeficiency.
[0472] In a specific embodiment, antibodies andlor compositions of the
invention are
administered to treat or ameliorate selective IgA deficiency.
[0473] In another specific embodiment, antibodies and/or compositions of the
invention are administered to treat or ameliorate ataxia-telangiectasia.
[0474] In another specific embodiment antibodies and/or compositions of the
invention are administered to treat or ameliorate common variable
immunodeficiency.
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[0475] In another specific embodiment, antibodies and/or compositions of the
invention are administered to treat or ameliorate X-linked agammaglobulinemia.
[0476] In another specific embodiment, antibodies and/or compositions of the
invention are administered to treat or ameliorate severe combined
immunodeficiency
(SLID).
[0477] In another specific embodiment, antibodies and/or compositions of the
invention are administered to treat or ameliorate Wiskott-Aldrich syndrome.
[0478] In another specific embodiment, antibodies and/or compositions of the
invention are administered to treat or ameliorate X-linked Ig deficiency with
hyper IgM.
[0479] As an agent to boost immunoresponsiveness among individuals having an
acquired loss of B cell function. Conditions resulting in an acquired loss of
B cell
function that may be ameliorated or treated by administering antibodies and/or
compositions of the invention include, but are not limited to, HIV Infection,
All~S, bone
marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
[0480] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent to boost immunoresponsiveness among individuals
having a
temporary immune deficiency. Conditions resulting in a temporary immune
deficiency
that may be ameliorated or treated by administering antibodies and/or
compositions of the
invention include, but are not limited to, recovery from viral infections
(e.g., influenza),
conditions associated with malnutrition, recovery from infectious
mononucleosis, or
conditions associated with stress, recovery from measles, recovery from blood
transfusion,
recovery from surgery.
[0481] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a regulator of antigen presentation by monocytes,
dendritic cells, T
cells and/or B-cells. In one embodiment, antibody polypeptides or
polynucleotides
enhance antigen presentation or antagonize antigen presentation in vitro or in
vivo.
Moreover, in related embodiments, this enhancement or antagonization of
antigen
presentation may be useful in anti-tumor treatment or to modulate the immune
system.
[0482] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a mediator of mucosal immune responses. The expression
of BLyS
on monocytes, the expression of BLyS receptor on B cells, and the
responsiveness of B
cells to BLyS suggests that it may be involved in exchange of signals between
B cells and
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monocytes or their differentiated progeny. This activity is in many ways
analogous to the
CD40-CD154 signalling between B cells and T cells. Anti-BLyS antibodies and
compositions of the invention may therefore be good regulators of T cell
independent
immune responses to environmental pathogens. In particular, the unconventional
B cell
populations (CD5+) that are associated with mucosal sites and responsible for
much of the
innate immunity in humans may respond to antibodies or compositions of the
invention
thereby enhancing or inhibiting individual's immune status.
[0483] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an agent to direct an individual's immune system towards
development of a humoral response (i.e. TH2) as opposed to a TH1 cellular
response.
[0484] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a means to induce tumor proliferation and thus make it
more
susceptible to anti-neoplastic agents. For example, multiple myeloma is a
slowly dividing
disease and is thus refractory to virtually all anti-neoplastic regimens. If
these cells were
forced to proliferate more rapidly, their susceptibility profile would likely
change.
[0485] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a monocyte cell specific binding protein to which
specific activators
or inhibitors of cell growth may be attached. The result would be to focus the
activity of
such activators or inhibitors onto normal, diseased, or neoplastic B cell
populations.
[0486] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a B cell specific binding protein to which specific
activators or
inhibitors of cell growth may be attached. The result would be to focus the
activity of
such activators or inhibitors onto normal, diseased, or neoplastic B cell
populations.
[0487] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a means of detecting monocytic cells by virtue of its
specificity.
This application may require labeling the protein with biotin or other agents
(e.g., as
described herein) to afford a means of detection.
[0488] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a means of detecting B-lineage cells by virtue of its
specificity. This
application may require labeling the protein with biotin or other agents
(e.g., as described
herein) to afford a means of detection.
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[0489] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a stimulator of B cell production in pathologies such as
AIDS,
chronic lymphocyte disorder and/or Common Variable immunodeficiency.
[0490] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as part of a monocyte selection device the function of
which is to
isolate monocytes from a heterogeneous mixture of cell types. Antibodies of
the invention
could be coupled to a solid support to which monocytes would then specifically
bind.
Unbound cells would be washed out and the bound cells subsequently eluted. A
non-
limiting use of this selection would be to allow purging of tumor cells from,
for example,
bone marrow or peripheral blood prior to transplant.
[0491] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as part of a B cell selection device the function of which
is to isolate B
cells from a heterogeneous mixture of cell types. Antibodies of the invention
(that do not
inhibit BLyS/BLyS Receptor interaction) binding soluble BLyS could be coupled
to a
solid support to which B cells would then specifically bind. Unbound cells
would be
washed out and the bound cells subsequently eluted. A non-limiting use of this
selection
would be to allow purging of tumor cells from, for example, bone marrow or
peripheral
blood prior to transplant.
[0492] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a therapy for generation and/or regeneration of lymphoid
tissues
following surgery, trauma or genetic defect.
[0493] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a gene-based therapy for genetically inherited disorders
resulting in
immuno-incompetence such as observed among SCID patients.
[0494] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as an antigen for the generation of antibodies to inhibit
or enhance
BLyS mediated responses.
[0495] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a means of activating monocytes/macrophages to defend
against
parasitic diseases that effect monocytes such as Leishmania.
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[0496] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as pretreatment of bone marrow samples prior to transplant.
Such
treatment would increase B cell representation and thus accelerate recovery.
[0497] In a specific embodiment, therapeutic or pharmaceutical compositions of
the
invention are used as a means of regulating secreted cytokines that are
elicited by BLyS
and/or BLyS receptor.
[0498] Antibody polypeptides or polynucleotides of the invention may be used
to
modulate IgE concentrations in vitro or in vivo.
[0499] Additionally, antibody polypeptides or polynucleotides of the invention
may be
used to treat, prevent, and/or diagnose IgE-mediated allergic reactions. Such
allergic
reactions include, but are not limited to, asthma, rhinitis, and eczema.
[0500] In a specific embodiment, antibody polypeptides or polynucleotides of
the
invention, are administered to treat, prevent, diagnose, and/or ameliorate
selective IgA
deficiency.
[0501] In another specific embodiment antibody polypeptides or polynucleotides
of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
ataxia-
telangiectasia.
[0502] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
common
variable immunodeficiency.
[0503] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
X-linked
agammaglobulinemia.
[0504] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
severe
combined immunodeficiency (SCID).
[0505] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
Wiskott-
Aldrich syndrome.
[0506] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
X-linked Ig
deficiency with hyper , IgM. In a specific embodiment antibody polypeptides or
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polynucleotides of the invention are administered to treat, prevent, diagnose,
and/or
ameliorate X-linked Ig deficiency with hyper IgM.
[0507] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, and/or diagnose chronic
myelogenous
leukemia, acute myelogenous leukemia, leukemia, hystiocytic leukemia,
monocytic
leukemia (e.g., acute monocytic leukemia), leukemic reticulosis, Shilling Type
monocytic
leukemia, and/or other leukemias derived from monocytes and/or monocytic cells
and/or
tissues.
[0508] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
monocytic
leukemoid reaction, as seen, for example, with tuberculosis.
[0509] In another specific embodiment, antibody polypeptides or
polynucleotides of
the invention are administered to treat, prevent, diagnose, and/or ameliorate
monocytic
leukocytosis, monocytic leukopenia, monocytopenia, and/or monocytosis.
[0510] In a specific embodiment, antibody polypeptides or polynucleotides of
the
invention are used to treat, prevent, detect, andlor diagnose monocyte
disorders and/or
diseases, and/or conditions associated therewith.
[0511] In a specific embodiment, antibody polypeptides or polynucleotides of
the
invention are used to treat, prevent, detect, andlor diagnose primary B
lymphocyte
disorders and/or diseases, and/or conditions associated therewith. In one
embodiment,
such primary B lymphocyte disorders, diseases, andlor conditions are
characterized by a
complete or partial loss of humoral immunity. Primary B lymphocyte disorders,
diseases,
and/or conditions associated therewith that are characterized by a complete or
partial loss
of humoral immunity and that may be prevented, treated, detected and/or
diagnosed with
compositions of the invention include, but are not limited to, X-Linked
Agammaglobulinemia (XLA), severe combined immunodeficiency disease (SCID), and
selective IgA deficiency.
[0512] In a preferred embodiment antibody polypeptides or polynucleotides of
the
invention are used to treat, prevent, and/or diagnose diseases or disorders
affecting or
conditions associated with any one or more of the various mucous membranes of
the body.
Such diseases or disorders include, but are not limited to, for example,
mucositis,
mucoclasis, mucocolitis, mucocutaneous leishmaniasis (such as, for example,
American
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leishmaniasis, leishmaniasis americana, nasopharyngeal leishmaniasis, and New
World
leishmaniasis), mucocutaneous lymph node syndrome (for example, Kawasaki
disease),
mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor, mucoepithelial
dysplasia, mucoid adenocarcinoma, mucoid degeneration, myxoid degeneration;
myxomatous degeneration; myxomatosis, mucoid medial degeneration (for example,
cystic medial necrosis), mucolipidosis (including, for example, mucolipidosis
I,
mucolipidosis II, mucolipidosis III, and mucolipidosis IV), mucolysis
disorders,
mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis (such as, for
example,
type I mucopolysaccharidosis (i.e., Hurler's syndrome), type IS
mucopolysaccharidosis
(i.e., Scheie's syndrome or type V rnucopolysaccharidosis), type II
mucopolysaccharidosis
(i.e., Hunter's syndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's
syndrome),
type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VI
mucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VII
mucopolysaccharidosis (i.e, mucopolysaccharidosis due to beta-glucuronidase
deficiency),
and mucosulfatidosis), mucopolysacchariduria, mucopurulent conjunctivitis,
mucopus,
mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virus
diarrhea), mucous
colitis (such as, for example, mucocolitis and myxomembranous colitis), and
mucoviscidosis (such as, for example, cystic fibrosis, cystic fibrosis of the
pancreas,
Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis,
and
viscidosis). In a highly preferred embodiment, antibody polypeptides or
polynucleotides
of the invention are used to treat, prevent, and/or diagnose mucositis,
especially as
associated with chemotherapy.
[0513] In a preferred embodiment, antibody polypeptides or polynucleotides of
the
invention are used to treat, prevent, and/or diagnose diseases or disorders
affecting or
conditions associated with sinusitis.
[0514] An additional condition, disease or symptom that can be treated,
prevented,
and/or diagnosed by antibody polypeptides or polynucleotides of the invention
is
osteomyelitis.
[0515] An additional condition, disease or symptom that can be treated,
prevented,
and/or diagnosed by antibody polypeptides or polynucleotides of the invention
is
endocarditis.
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[0516] All of the above described applications as they may apply to veterinary
medicine.
[0517] Antibody polypeptides or polynucleotides of the invention may be used
to
treat, prevent, and/or diagnose diseases and disorders of the pulmonary system
(e.g.,
bronchi such as, for example, sinopulmonary and bronchial infections and
conditions
associated with such diseases and disorders and other respiratory diseases and
disorders.
In specific embodiments, such diseases and disorders include, but are not
limited to,
bronchial adenoma, bronchial asthma, pneumonia (such as, e.g., bronchial
pneumonia,
bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive
pulmonary
disease (COPD), bronchial polyps, bronchiectasia (such as, e.g.,
bronchiectasia sicca,
cylindrical bronchiectasis, and saccular bronchiectasis), bronchiolar
adenocarcinoma,
bronchiolar carcinoma, bronchiolitis (such as, e.g., exudative bronchiolitis,
bronchiolitis
fibrosa obliterans, and proliferative bronchiolitis), bronchiolo-alveolar
carcinoma,
bronchitis asthma, bronchitis (such as, e.g., asth'matic bronchitis,
Castellani's bronchitis,
chronic bronchitis, croupous bronchitis, fibrinous bronchitis, hemorrhagic
bronchitis,
infectious avian bronchitis, obliterative bronchitis, plastic bronchitis,
pseudomembranous
bronchitis, putrid bronchitis, and verminous bronchitis), bronchocentric
granulomatosis,
bronchoedema, bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic
cyst,
broncholithiasis, bronchomalacia, bronchomycosis (such as, e.g.,
bronchopulmonary
aspergillosis), bronchopulmonary spirochetosis, hemorrhagic bronchitis,
bronchorrhea,
bronchospasrn, bronchostaxis, bronchostenosis, Biot's respiration, bronchial
respiration,
Kussmaul respiration, Kussmaul-Kien respiration, respiratory acidosis,
respiratory
alkalosis, respiratory distress syndrome of the newborn, respiratory
insufficiency,
respiratory scleroma, respiratory syncytial virus, and the like.
[0518] In a specific embodiment, antibody polypeptides or polynucleotides of
the
invention are used to treat, prevent, and/or diagnose chronic obstructive
pulmonary
disease (COPD).
[0519] In another embodiment, antibody polypeptides or polynucleotides of the
invention are used to treat, prevent, and/or diagnose fibroses and conditions
associated
with fibroses, including, but not limited to, cystic fibrosis (including such
fibroses as
cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease
of the
pancreas, mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathic
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retroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis,
nodular
subepidermal fibrosis, pericentral fibrosis, perimuscular fibrosis, pipestem
fibrosis,
replacement fibrosis, subadventitial fibrosis, and Symmers' clay pipestem
fibrosis.
[0520] In another embodiment, therapeutic or pharmaceutical compositions of
the
invention are administered to an animal to treat, prevent or ameliorate
infectious diseases.
Infectious diseases include diseases associated with yeast, fungal, viral and
bacterial
infections. Viruses causing viral infections which can be treated or prevented
in
accordance with this invention include, but are not limited to, retroviruses
(e.g., human
T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency
virus
(HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types I and II,
Epstein-Barr virus,
HHV6-HHVB, and cytomegalovirus), arenavirues (e.g., lassa fever virus),
paramyxoviruses (e.g., morbillivirus virus, human respiratory syncytial virus,
mumps, and
pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses,
filoviruses
(e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellow fever
virus, and
Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B viruses
(HBV)),
orthomyoviruses (e.g., influenza viruses A, B and C), papovaviruses (e.g.,
papillomavirues), picornaviruses (e.g., rhinoviruses, enteroviruses and
hepatitis A viruses),
poxviruses, reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus),
rhabdoviruses
(e.g., rabies virus). Microbial pathogens causing bacterial infections
include, but are not
limited to, Streptococcus pyogeraes, Streptococcus pneumorziae, Neisseria
gonorrhoea,
Neisseria rneningitidis, Corynebacterium diphtheriae , Clostridium botulinum,
Clostridium perfringens, Clostridium tetani, Haenzophilus influenzae,
Klebsiella
pneurnoniae, Klebsiella ozaenae, Klebsiella rhirzoscleromotis, Staphylococcus
aureus,
Vibrzo cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter
(Vibrio)
fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus,
Edwardsiella tarda,
Yersznia eraterocolitica, Yersinia pestis, Yersirzia pseudotuberculosis,
Shigella dyserzteriae,
Shigella flexneri, Shigella sonnei, Salrnonella typhimurium, Treporzema
pallidurn,
Treponerna pertenue, Treponema carateneum, Borrelia virzcentii, Borrelia
burgdorfer-i,
Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasnza gondii,
Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis,
Brucella
melitensis, Mycoplasnza spp., Rickettsia prowazeki, Rickettsia tsutsugunzushi,
Clzlamydia
spp., and Helicobacter pylori.
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Gene Therapy
[0521] In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or prevent a
disease or disorder associated with aberrant expression and/or activity of
BLyS and/or its
receptor, by way of gene therapy. Gene therapy refers to therapy performed by
the
administration to a subject of an expressed or expressible nucleic acid. In
this
embodiment of the invention, the nucleic acids produce their encoded protein
that
mediates a therapeutic effect.
[0522] Any of the methods for gene therapy available in the art can be used
according
to the present invention. Exemplary methods are described below.
[0523] For general reviews of the methods of gene therapy, see Goldspiel et
al.,
Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3: ~7-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
(1993);
May, TIBTECH 1 l(5):ISS-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in Ausubel et al.
(eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and
Kriegler,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0524] In a preferred aspect, a composition of the invention comprises, or
alternatively
consists of, nucleic acids encoding an antibody, said nucleic acids being part
of an
expression vector that expresses the antibody or fragments or chimeric
proteins or heavy
or light chains thereof in a suitable host. In particular, such nucleic acids
have promoters,
preferably heterologous promoters, operably linked to the antibody coding
region, said
promoter being inducible or constitutive, and, optionally, tissue-specific. In
another
particular embodiment, nucleic acid molecules are used in which the antibody
coding
sequences and any other desired sequences are flanked by regions that promote
homologous recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the antibody encoding nucleic acids (Koller and
Smithies,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature
342:435-438
(1989). In specific embodiments, the expressed antibody molecule is an scFv;
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alternatively, the nucleic acid sequences include sequences encoding both the
heavy and
light chains, or fragments or variants thereof, of an antibody.
[0525] Delivery of the nucleic acids into a patient may be either direct, in
which case
the patient is directly exposed to the nucleic acid or nucleic acid- carrying
vectors, or
indirect, in which case, cells are first transformed with the nucleic acids in
vitro, then
transplanted into the patient. These two approaches are known, respectively,
as in vivo or
ex vivo gene therapy.
[0526] In a specific embodiment, the nucleic acid sequences are directly
administered
in vivo, where it is expressed to produce the encoded product. This can be
accomplished
by any of numerous methods known in the art, e.g., by constructing them as
part of an
appropriate nucleic acid expression vector and administering it so that they
become
intracellular, e.g., by infection using defective or attenuated retrovirals or
other viral
vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA,
or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids or
cell-surface receptors or transfecting agents, encapsulation in liposomes,
microparticles, or
microcapsules, or by administering them in linkage to a peptide which is known
to enter
the nucleus, by administering it in linkage to a ligand subject to receptor-
mediated
endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which
can be
used to target cell types specifically expressing the receptors), etc. In
another
embodiment, nucleic acid-ligand complexes can be formed in which the ligand
comprises
a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to
avoid
lysosomal degradation. In yet another embodiment, the nucleic acid can be
targeted ifa
vivo for cell specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT
Publications WO 92/06 180; WO 92/22635; W092/203 16; W093/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly and
incorporated within
host cell DNA for expression, by homologous recombination (Koller and
Smithies, Proc.
Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[0527] In a specific embodiment, viral vectors that contains nucleic acid
sequences
encoding an antibody of the invention or fragments or variants thereof are
used. For
example, a retroviral vector can be used (see Miller et al., Meth. Enzymol.
217:581-599
(1993)). These retroviral vectors contain the components necessary for the
correct
packaging of the viral genome and integration into the host cell DNA. The
nucleic acid
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sequences encoding the antibody to be used in gene therapy are cloned into one
or more
vectors, which facilitates delivery of the gene into a patient. More detail
about retroviral
vectors can be found in Boesen et al., Biotherapy 6:29 1-302 (1994), which
describes the
use of a retroviral vector to deliver the mdr 1 gene to hematopoietic stem
cells in order to
make the stem cells more resistant to chemotherapy. Other references
illustrating the use
of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-651(1994);
Klein et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene
Therapy
4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.
3:110-
114 (1993).
[0528] Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a mild
disease. Other targets for adenovirus-based delivery systems are liver, the
central nervous
system, endothelial cells, and muscle. Adenoviruses have the advantage of
being capable
of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in
Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based gene
therapy. Bout
et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus
vectors to
transfer genes to the respiratory epithelia of rhesus monkeys. Other instances
of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-
434
(1991); Rosenfeld et al., Cell 68:143- 155 (1992); Mastrangeli et al., J.
Clin. Invest.
91:225-234 (1993); PCT Publication W094112649; and Wang, et al., Gene Therapy
2:775-
783 (1995). In a preferred embodiment, adenovirus vectors are used.
[0529] Adeno-associated virus (AAV) has also been proposed for use in gene
therapy
(Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
[0530] Another approach to gene therapy involves transferring a gene to cells
in tissue
culture by such methods as electroporation, lipofection, calcium phosphate
mediated
transfection, or viral infection. Usually, the method of transfer includes the
transfer of a
selectable marker to the cells. The cells are then placed under ,selection to
isolate those
cells that have taken up and are expressing the transferred gene. Those cells
are then
delivered to a patient.
[0531] In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be carried
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out by any method known in the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector containing
the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer,
microcell-
mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known
in the art
for the introduction of foreign genes into cells (see, e.g., Loeffler and
Behr, Meth.
Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);
Clin.
Pharma. Ther. 29:69-92m (1985)) and may be used in accordance with the present
invention, provided that the necessary developmental and physiological
functions of the
recipient cells are not disrupted. The technique should provide for the stable
transfer of
the nucleic acid to the cell, so that the nucleic acid is expressible by the
cell and preferably
heritable and expressible by its cell progeny.
[0532] The resulting recombinant cells can be delivered to a patient by
various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
envisioned for use depends on the desired effect, patient state, etc., and can
be determined
by one skilled in the art.
[0533] Cells into which a nucleic acid can be introduced for purposes of gene
therapy
encompass any desired, available cell type, and include but are not limited to
epithelial
cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells
such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,
eosinophils, megakaxyocytes, granulocytes; various stem or progenitor cells,
in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow,
umbilical
cord blood, peripheral blood, fetal liver, etc.
[0534] In a preferred embodiment, the cell used for gene therapy is autologous
to the
patient.
[0535] In an embodiment in which recombinant cells are used in gene therapy,
nucleic
acid sequences encoding an antibody or fragment thereof are introduced into
the cells such
that they are expressible by the cells or their progeny, and the recombinant
cells are then
administered in vivo for therapeutic effect. In a specific embodiment, stem or
progenitor
cells are used. Any stem and/or progenitor cells which can be isolated and
maintained ifa
vitro can potentially be used in accordance with this embodiment of the
present invention
(see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 7 1:973-985
(1992);
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Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo
Clinic Proc.
61:771 (1986)).
[0536] In a specific embodiment, the nucleic acid to be introduced for
purposes of
gene therapy comprises an inducible promoter operably linked to the coding
region, such
that expression of the nucleic acid is controllable by controlling the
presence or absence of
the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Utility of a Com osition
[0537] The'compounds of the invention are preferably tested in vitro, and then
in vivo
for the desired therapeutic or prophylactic activity, prior to use in humans.
For example,
in vitro assays which can be used to determine whether administration of a
specific
antibody or composition of the present invention is indicated, include ih
vitro cell culture
assays in which a patient tissue sample is grown in culture, and exposed to or
otherwise
administered an antibody or composition of the present invention, and the
effect of such
an antibody or composition of the present invention upon the tissue sample is
observed. In
various specific embodiments, ifa vitro assays can be carried out with
representative cells
of cell types involved in a patient's disorder, to determine if an antibody or
composition of
the present invention has a desired effect upon such cell types. Preferably,
the antibodies
or compositions of the invention are also tested in in vitro assays and animal
model
systems prior to administration to humans.
[0538] Antibodies or compositions of the present invention for use in therapy
can be
tested for their toxicity in suitable animal model systems, including but not
limited to rats,
mice, chicken, cows, monkeys, and rabbits. For i~z vivo testing of an antibody
~ or
composition's toxicity any animal model system known in the art may be used.
[0539] Efficacy in treating or preventing viral infection may be demonstrated
by
detecting the ability of an antibody or composition of the invention to
inhibit the
replication of the virus, to inhibit transmission or prevent the virus from
establishing itself
in its host, or to prevent, ameliorate or alleviate the symptoms of disease a
progression.
The treatment is considered therapeutic if there is, for example, a reduction
in viral load,
amelioration of one or more symptoms, or a decrease in mortality and/or
morbidity
following administration of an antibody or composition of the invention.
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[0540] Antibodies or compositions of the invention can be tested for the
ability to
induce the expression of cytokines such as IFN-y, by contacting cells,
preferably human
cells, with an antibody or composition of the invention or a control antibody
or control
composition and determining the ability of the antibody or composition of the
invention to
induce one or more cytokines. Techniques known to those of skill in the art
can be used to
measure the level of expression of cytokines. For example, the level of
expression of
cytokines can be measured by analyzing the level of RNA of cytokines by, for
example,
RT-PCR and Northern blot analysis, and by analyzing the level of cytokines by,
for
example, immunoprecipitation followed by western blot analysis and ELISA. In a
preferred embodiment, a compound of the invention is tested for its ability to
induce the
expression of IFN-y.
[0541] Antibodies or compositions of the invention can be tested for their
ability to
modulate the biological activity of immune cells by contacting immune cells,
preferably
human immune cells (e.g., T-cells, B-cells, and Natural Filler cells), with an
antibody or
composition of the invention or a control compound and determining the ability
of the
antibody or composition of the invention to modulate (i.e, increase or
decrease) the
biological activity of immune cells. The ability of an antibody or composition
of the
invention to modulate the biological activity of immune cells can be assessed
by detecting
the expression of antigens, detecting the proliferation of immune cells (i.e.,
B-cell
proliferation), detecting the activation of signaling molecules, detecting the
effector
function of immune cells, or detecting the differentiation of immune cells.
Techniques
known to those of skill in the art can be used for measuring these activities.
For example,
cellular proliferation can be assayed by 3H-thymidine incorporation assays and
trypan
blue cell counts. Antigen expression can be assayed, for example, by
immunoassays
including, but not limited to, competitive and non-competitive assay systems
using
techniques such as western blots, immunohistochemistry radioimmunoassays,
ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays,
agglutination assays, complement-fixation assays, immunoradiometric assays,
fluorescent
immunoassays, protein A immunoassays and FACS analysis. The activation of
signaling
molecules can be assayed, for example, by kinase assays and electrophoretic
shift assays
(EMSAs). In a preferred embodiment, the ability of an antibody or composition
of the
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invention to induce B-cell proliferation is measured. In another preferred
embodiment, the
ability of an antibody or composition of the invention to modulate
immunoglobulin
expression is measured.
[0542] Antibodies or compositions of the invention can be tested for their
ability to
reduce tumor formation in in vitro, ex vivo and i~ vivo assays. Antibodies or
compositions
of the invention can also be tested for their ability to inhibit viral
replication or reduce
viral load in ih vitro and in vivo assays. Antibodies or compositions of the
invention can
also be tested for their ability to reduce bacterial numbers in i~c vitro and
in vivo assays
known to those of skill in the art. Antibodies or compositions of the
invention can also be
tested for their ability to alleviate of one or more symptoms associated with
cancer, an
immune disorder (e.g., an inflammatory disease), a neurological disorder or an
infectious
disease. Antibodies or compositions of the invention can also be tested for
their ability to
decrease the time course of the infectious disease. Further, antibodies or
compositions of
the invention can be tested for their ability to increase the survival period
of animals
suffering from disease or disorder, including cancer, an immune disorder or an
infectious
disease. Techniques known to those of skill in the art can be used to analyze
the function
of the antibodies or compositions of the invention ih vivo.
Therapeutic/Prophylactic Compositions and Administration
[0543] The invention provides methods of treatment, inhibition and prophylaxis
by
administration to a subject of an effective amount of antibody (or fragment or
variant
thereof) or pharmaceutical composition of the invention, preferably an
antibody of the
invention. In a preferred aspect, an antibody or fragment or variant thereof
is substantially
purified (i.e., substantially free from substances that limit its effect or
produce undesired
side-effects). The subject is preferably an animal, including but not limited
to, animals
such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and
most preferably a human.
[0544] Formulations and methods of administration that can be employed when
the
compound comprises a nucleic acid or an immunoglobulin as described above;
additional
appropriate formulations and routes of administration can be selected from
among those
described herein below.
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[0545] Various delivery systems are known and can be used to administer
antibody or
fragment or variant thereof of the invention, e.g., encapsulation in
liposomes,
microparticles, microcapsules, recombinant cells capable of expressing the
antibody or
antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.
Biol. Chem.
262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral
or other
vector, etc. Methods of introduction include, but are not limited to,
intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,
epidural, and oral
routes. The compositions may be administered by any convenient route, for
example by
infusion or bolus injection, by absorption through epithelial or mucocutaneous
linings
(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together
with other biologically active agents. Administration can be systemic or
local. In addition,
it may be desirable to introduce the pharmaceutical compositions of the
invention into the
central nervous system by any suitable route, including intraventricular and
intrathecal
injection; intraventricular injection may be facilitated by an
intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary
administration
can also be employed, e.g., by use of an inhaler or nebulizer, and formulation
with an
aerosolizing agent.
[0546] In a preferred embodiment the antibody of the invention is formulated
in 10
mM sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (~
0.3). In
another preferred embodiment, the antibody of the invention is formulated in
10 mM
sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (~
0.3) for
intravenous administration.
[0547] In a specific embodiment, it may be desirable to administer the
pharmaceutical
compositions of the invention locally to the area in need of treatment; this
may be
achieved by, for example, and not by way of limitation, local infusion during
surgery,
topical application, e.g., in conjunction with a wound dressing after surgery,
by injection,
by means of a catheter, by means of a suppository, or by means of an implant,
said implant
being of a porous, non-porous, or gelatinous material, including membranes,
such as
sialastic membranes, or fibers. Preferably, when administering a protein,
including an
antibody, of the invention, care must be taken to use materials to which the
protein does
not absorb.
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[0548] In another embodiment, the composition can be delivered in a vesicle,
in
particular a liposome (see Larger, Science 249:1527-1533 (1990); Treat et al.,
in
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler
(eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 3 17-
327; see
generally ibid.).
[0549] In yet another embodiment, the composition can be delivered in a
controlled
release system. In one embodiment, a pump may be used (see Larger, supra;
Sefton, CRC
Crit. Ref. Biomed. Eng. 14:20 1 (1987); Buchwald et al., Surgery 88:507
(1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric
materials can
be used (see Medical Applications of Controlled Release, Larger and Wise
(eds.), CRC
Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug
Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and
Peppas,
J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,
Science
228:190 (1985); During et al., Ann. Neurol. 25:35 1 (1989); Howard et al.,
J.Neurosurg. 7
1:105 (1989)). In yet another embodiment, a controlled release system can be
placed in
proximity of the therapeutic target, i.e., the brain, thus requiring only a
fraction of the
systemic dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra,
vol. 2, pp. 115-138 (1984)).
[0550] Other controlled release systems are discussed in the review by Larger
(Science 249:1527-1533 (1990)).
[0551] In a specific embodiment where the composition of the invention is a
nucleic
acid encoding a protein, the nucleic acid can be administered in vivo to
promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic acid
expression vector and administering it so that it becomes intracellular, e.g.,
by use of a
retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or
by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids or
cell-surface receptors or transfecting agents, or by administering it in
linkage to a
homeobox- like peptide which is known to enter the nucleus (see e.g., Joliot
et al., Proc.
Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid
can be
introduced intracellularly and incorporated within host cell DNA for
expression, by
homologous recombination.
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[0552] The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of an antibody or a
fragment
thereof, and a pharmaceutically acceptable carrier. In a specific embodiment,
the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a
state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids, such as
water and oils,
including those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil,
soybean oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the
pharmaceutical composition is administered intravenously. Saline solutions and
aqueous
dextrose and glycerol solutions can also be employed as liquid carriers,
particularly for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the
like. The composition, if desired, can also contain minor amounts of wetting
or
emulsifying agents, or pH buffering agents. These compositions can take the
form of
solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-
release
formulations and the like. The composition can be formulated as a suppository,
with
traditional binders and carriers such as triglycerides. Oral formulation can
include
standard carriers such as pharmaceutical grades of mannitol, lactose, starch,
magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable
pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences"
by E.W.
Martin. Such compositions will contain a therapeutically effective amount of
the antibody
or fragment thereof, preferably in purified form, together with a suitable
amount of carrier
so as to provide the form for proper administration to the patient. The
formulation should
suit the mode of administration.
[0553] In a preferred embodiment, the composition is formulated in accordance
with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compositions for intravenous
administration
are solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may
also include a solubilizing agent and a local anesthetic such as lignocamne to
ease pain at
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the site of the injection. Generally, the ingredients are supplied either
separately or mixed
together in unit dosage form, for example, as a dry lyophilized powder or
water free
concentrate in a hermetically sealed container such as an ampoule or sachette
indicating
the quantity of active agent. Where the composition is to be administered by
infusion, it
can be dispensed with an infusion bottle containing sterile pharmaceutical
grade water or
saline. Where the composition is administered by injection, an ampoule of
sterile water for
injection or saline can be provided so that the ingredients rnay be mixed
prior to
administration.
[0554] The compositions of the invention can be formulated as neutral or salt
forms.
Pharmaceutically acceptable salts include those formed with anions such as
those derived
from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with
cations such as those derived from sodium, potassium, ammonium, calcium,
ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0555] The amount of the composition of the invention which will be effective
in the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
expression and/or activity of a polypeptide of the invention can be determined
by standard
clinical techniques. In addition, in vitro assays may optionally be employed
to help
identify optimal dosage ranges. The precise dose to be employed in the
formulation will
also depend on the route of administration, and the seriousness of the disease
or disorder,
and should be decided according to the judgment of the practitioner and each
patient's
circumstances. Effective doses may be extrapolated from dose-response curves
derived
from ih vitro or animal model test systems.
[0556] For antibodies, the dosage administered to a patient is typically 0.1
mg/kg to
100 mg/kg of the patient's body weight. Preferably, the dosage administered to
a patient
is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more
preferably 1 mg/kg
to 10 mg/kg of the patient's body weight. In preferred embodiments, a dose of
l, 4, 10, or
20 mg/kg is administered intravenously to a patient. Generally, human
antibodies have a
longer half-life within the human body than antibodies from other species due
to the
immune response to the foreign polypeptides. Thus, lower dosages of human
antibodies
and less frequent administration is often possible. Further, the dosage and
frequency of
administration of therapeutic or pharmaceutical compositions of the invention
may be
242
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