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CA 02382659 2002-O1-29
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Dendritic Enriched Secreted Lymphocyte Activation Molecule
Field of the Invention
The present invention relates to a novel human gene encoding a polypeptide
which is
a member of the Secreted Lymphocyte Activation Molecule (SLAM) family. More
S specifically, the present invention relates to a polynucleotide encoding a
novel human
polypeptide named Dendritic Enriched Secreted Lymphocyte Activation Molecule,
or "D-
SLAM." This invention also relates to D-SLAM polypeptides, as well as vectors,
host cells,
antibodies directed to D-SLAM polypeptides, and the recombinant methods for
producing the
same. Also provided are diagnostic methods for detecting disorders related to
the immune
system, and therapeutic methods for treating, diagnosing, detecting, and/or
preventing such
disorders. The invention further relates to screening methods for identifying
agonists and
antagonists of D-SLAM activity.
Background of the Invention
A member of the immunoglobulin gene superfamily, SLAM is rapidly induced after
activation of naive T- and B-cells. (Cocks, B.G., "A Novel Receptor Involved
in T-Cell
Activation," Nature 376:260-263 (1995); Aversa, G., "Engagement of the
Signaling
Lymphocytic Activation Molecule (SLAM) on Activated T Cells Results in Il-2-
Independent,
Cyclosporin A-Sensitive T Cell Proliferation and IFN-gamma Production," J.
Immun. 4036-
4044 (1997).) A multifunctional 70 kDa glycoprotein, SLAM causes proliferation
and
differentiation of immune cells. (Punnonen, J., "Soluble and Membrane-bound
Forms of
Signaling Lymphocytic Activation Molecule (SLAM) Induce Proliferation and Ig
Synthesis
by Activated Human B Lymphocytes," J. Exp. Med. 185:993-1004 (1997).) To
elicit an
immune response, both a secreted form of SLAM, as well as a membrane bounded
SLAM,
are thought to interact.
It is also known that dendritic cells (DC) are the principal antigen
presenting cells
involved in primary immune responses; their major function is to obtain
antigen in tissues,
migrate to lymphoid organs, and activate T cells. (Mohamadzadeh, M. et al., J.
Immunol.
156: 3102-3106 (1996).) In fact, DC are usually the first immune cells to
arrive at sites of
inflammation on mucous membranes. (See, e.g., Weissman, D. et al., J. Immunol.
155:4111 -
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2
4117 (1995).) DC are also known to directly interact with B cells. There is a
constant need to
identify new polypeptide factors which may mediate interactions between DC and
T cells
and/or B cells, leading to the activation and/or proliferation of immune
cells. To date,
however, SLAM molecules have not been identified on DC cells.
Thus, there is a need for polypeptides that affect the proliferation,
activation, survival,
and/or differentiation of immune cells, such as T- and B-cells, since
disturbances of such
regulation may be involved in disorders relating to immune system. Therefore,
there is a
need for identification and characterization of such human polypeptides which
can play a role
in detecting, preventing, ameliorating or correcting such disorders.
l0 Summary of the Invention
The present invention relates to a novel polynucleotide and the encoded
polypeptide
of D-SLAM. Moreover, the present invention relates to vectors, host cells,
antibodies, and
recombinant methods for producing the polypeptides and polynucleotides. Also
provided are
diagnostic methods for detecting and/or diagnosing disorders relates to the
polypeptides, and
therapeutic methods for treating and/or preventing such disorders. The
invention further
relates to screening methods for identifying binding partners of D-SLAM.
In accordance with one embodiment of the present invention, there is provided
a
novel mature D-SLAM polypeptide, as well as biologically active and
diagnostically or
therapeutically useful fragments, analogs and derivatives thereof.
In accordance with another embodiment of the present invention, there are
provided
isolated nucleic acid molecules encoding human D-SLAM, including mRNAs, DNAs,
cDNAs, genomic DNAs as well as analogs and biologically active and
diagnostically or
therapeutically useful fragments and derivatives thereof.
The present invention provides isolated nucleic acid molecules comprising, or
alternatively, consisting of, a polynucleotide encoding a cytokine that are
structurally similar
to Secreted Lymphocyte Activation Molecules (SLAM) and related cytokines and
have
similar biological effects and activities. This cytokine is named D-SLAM and
the invention
includes D-SLAM polypeptides having at least a portion of the amino acid
sequence in
Figures 1A, 1B, 1C, and 1D (SEQ ID N0:2) or amino acid sequence encoded by the
cDNA
clone (HDPJ039) deposited on February 6, 1998 assigned ATCC number 209623. The
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3
nucleotide sequence determined by sequencing the deposited D-SLAM clone, which
is shown
in Figures 1A, 1B, 1C, and 1D (SEQ ID NO:1), contains an open reading frame
encoding a
complete polypeptide of 285 amino acid residues including an N-terminal
methionine (i.e.,
amino acid residues 1-285 of SEQ ID N0:2), a predicted signal peptide of about
22 amino
acid residues (i.e., amino acid residues 1-22 of SEQ ID N0:2), a predicted
mature form of
about 263 amino acids (i.e., amino acid residues 23-285 of SEQ ID N0:2), and a
deduced
molecular weight for the complete protein of about 34.2 kDa.
Thus, one embodiment of the invention provides an isolated nucleic acid
molecule
comprising, or alternatively consisting of, a polynucleotide having a
nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence encoding a
full-length D
SLAM polypeptide having the complete amino acid sequence in Figures 1A, 1B,
1C, and 1D
(SEQ ID N0:2) . or as encoded by the cDNA clone contained in the deposit
having ATCC
accession number 209623; (b) a nucleotide sequence encoding the predicted
extracellular
domain of the D-SLAM polypeptide having the amino acid sequence at positions
23 to 232 in
Figures 1A, 1B, 1C, and 1D (SEQ ID N0:2) or as encoded by the clone contained
in the
deposit having ATCC accession number 209623; (c) a nucleotide sequence
encoding a
fragment of the polypeptide of (b) having D-SLAM functional activity (e.g.,
biological
acitivity); (d) a nucleotide sequence encoding a polypeptide comprising the D-
SLAM
intracellular domain (predicted to constitute amino acid residues from about
256 to about 285
in Figures 1A, 1B, 1C, and 1D (SEQ ID N0:2)) or as encoded by the clone
contained in the
deposit having ATCC accession number 209623; (e) a nucleotide sequence
encoding a
polypeptide comprising the D-SLAM transmembrane domain (predicted to
constitute amino
acid residues from about 233 to about 255 in Figures 1A, 1B, 1C, and 1D (SEQ
ID N0:2) or
as encoded by the cDNA clone contained in the deposit having ATCC accession
number
209623; (f) a nucleotide sequence encoding a soluble D-SLAM polypeptide having
the
extracellular and intracellular domains but lacking the transmembrane domain;
and (g) a
nucleotide sequence complementary to any of the nucleotide sequences in (a),
(b), (c), (d), (e)
or (f) above.
Further embodiments of the invention include isolated nucleic acid molecules
that
comprise, or alternatively consist of, a polynucleotide having a nucleotide
sequence at least
80%, 85% or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or
99%
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4
identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f)
or (g) above, or a
polynucleotide which hybridizes under stringent hybridization conditions to a
polynucleotide
in (a), (b), (c), (d), (e), (f) or (g) above. This polynucleotide which
hybridizes does not
hybridize under stringent hybridization conditions to a polynucleotide having
a nucleotide
sequence consisting of only A residues or of only T residues.
In additional embodiments, the nucleic acid molecules of the invention
comprise, or
alternatively consist of, a polynucleotide which encodes the amino acid
sequence of an
epitope-bearing portion of a D-SLAM polypeptide having an amino acid sequence
in (a), (b),
(c), (d), (e), (f) or (g) above. A further nucleic acid embodiment of the
invention relates to an
isolated nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide
which encodes the amino acid sequence of a D-SLAM polypeptide having an amino
acid
sequence which contains at least one amino acid addition, substitution, and/or
deletion but
not more than 50 amino acid additions, substitutions and/or deletions, even
more preferably,
not more than 40 amino acid additions, substitutions, and/or deletions, still
more preferably,
not more than 30 amino acid additions, substitutions, and/or deletions, and
still even more
preferably, not more than 20 amino acid additions, substitutions, and/or
deletions. Of course,
in order of ever-increasing preference, it is highly preferable for a
polynucleotide which
encodes the amino acid sequence of a D-SLAM polypeptide to have an amino acid
sequence
which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 or 1-100, 1-50, 1-
25, 1-20, 1-15, 1-
10, or 1-5 amino acid additions, substitutions and/or deletions. Conservative
substitutions are
preferable.
The present invention also relates to recombinant vectors, which include the
isolated
nucleic acid molecules of the present invention, and to host cells containing
the recombinant
vectors, as well as to methods of making such vectors and host cells and for
using them for
production of D-SLAM polypeptides by recombinant techniques.
In accordance with a further embodiment of the present invention, there is
provided a
process for producing such polypeptides by recombinant techniques comprising
culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a D-SLAM
nucleic acid
sequence of the invention, under conditions promoting expression of said
polypeptide and
subsequent recovery of said polypeptide.
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The invention further provides an isolated D-SLAM polypeptide comprising, or
alternatively consisting of, an amino acid sequence selected from the group
consisting of: (a)
the amino acid sequence of the full-length D-SLAM polypeptide having the
complete amino
acid sequence shown in Figures 1A, 1B, 1C, and 1D (i.e., positions 1-285 of
SEQ ID N0:2)
5 or as encoded by the cDNA plasmid contained in the deposit having ATCC
accession number
209623; (b) the amino acid sequence of the full-length D-SLAM polypeptide
having the
complete amino acid sequence shown in SEQ ID N0:2 excepting the N-terminal
methionine
(i.e., positions 2 to 285 of SEQ ID N0:2); (c) a fragment of the polypeptide
of (b) having D-
SLAM functional activity (e.g., biological activity); (d) the amino acid
sequence of the
predicted extracellular domain of the D-SLAM polypeptide having the amino acid
sequence
at positions 23 to 232 in Figures 1A, 1B, 1C, and 1D (SEQ ID N0:2) or as
encoded by the
cDNA plasmid contained in the deposit having ATCC accession number 209623; (e)
the
amino acid sequence of the D-SLAM intracellular domain (predicted to
constitute amino acid
residues from about 256 to about 285 in Figures 1A, 1B, 1C, and 1D (SEQ ID
N0:2)) or as
encoded by the cDNA plasmid contained in the deposit having ATCC accession
number
209623; (f) the amino acid sequence of the D-SLAM transmembrane domain
(predicted to
constitute amino acid residues from about 233 to about 255 in Figures 1A, 1B,
1C, and 1D
(SEQ ID N0:2)) or as encoded by the cDNA plasmid contained in the deposit
having ATCC
accession number 209623; (g) the amino acid sequence of the soluble D-SLAM
polypeptide
having the extracellular and intracellular domains but lacking the
transmembrane domain,
wherein each of these domains is defined above; and (h) fragments of the
polypeptide of (a),
(b), (c), (d), (e), (f) or (g). The polypeptides of the present invention also
include
polypeptides having an amino acid sequence at least 80% identical, more
preferably at least
85% or 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%
identical to
those described in (a), (b), (c), (d), (e), (f) or (g) above, as well as
polypeptides having an
amino acid sequence with at least 80%, 85%, or 90% similarity, and more
preferably at least
95% similarity, to those above. Additional embodiments of the invention
relates to
polypeptides which comprise, or alternatively consist of, the amino acid
sequence of an
epitope-bearing portion of a D-SLAM polypeptide having an amino acid sequence
described
in (a), (b), (c), (d), (e), (f), (g) or (h) above. Polypeptides having the
amino acid sequence of
an epitope-bearing portion of a D-SLAM polypeptide of the invention include
portions of
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6
such polypeptides with at least 4, at least 5, at least 6, at least 7, at
least 8, and preferably 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 more preferably at least
about 30 amino acids
to about 50 amino acids, although epitope-bearing polypeptides of any length
up to and
including the entire amino acid sequence of a polypeptide of the invention
described above
also are included in the invention.
The present invention also encompasses the above polynucleotide sequences
fused to
a heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotides and
nucleic acid molecules are also encompassed by the invention.
Certain non-exclusive embodiments of the invention relate to a polypeptide
which has
the amino acid sequence of an epitope-bearing portion of a D-SLAM polypeptide
having an
amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h) or (i)
above. In other
embodiments, the invention provides an isolated antibody that binds
specifically (i.e.,
uniquely) to a D-SLAM polypeptide having an amino acid sequence described in
(a), (b), (c),
(d), (e), (f), (g), (h) or (i) above.
The invention further provides methods for isolating antibodies that bind
specifically
(i.e., uniquely) to a D-SLAM polypeptide having an amino acid sequence as
described herein.
Such antibodies are useful diagnostically or therapeutically as described
below.
The invention also provides for pharmaceutical compositions comprising soluble
D-
SLAM polypeptides, particularly human D-SLAM polypeptides, and/or anti-D-SLAM
antibodies which may be employed, for instance, to treat, prevent, prognose
and/or diagnose
tumor and tumor metastasis, infections by bacteria, viruses and other
parasites,
immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmune
diseases, graft
versus host disease, stimulate peripheral tolerance, destroy some transformed
cell lines,
mediate cell activation, survival and proliferation, to mediate immune
regulation and
inflammatory responses, and to enhance or inhibit immune responses.
In certain embodiments, soluble D-SLAM polypeptides of the invention, or
antagonists thereof, are administered, to treat, prevent, prognose and/or
diagnose an
immunodeficiency (e.g., severe combined immunodeficiency (SCID)-X linked, SCID-
autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked
agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-
linked
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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) or conditions
associated
with an immunodeficiency.
In a specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention,
or antagonists thereof, is administered to treat, prevent, prognose and/or
diagnose common
variable immunodeficiency.
In a specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention,
or antagonists thereof, is administered to treat, prevent, prognose and/or
diagnose X-linked
agammaglobulinemia.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent, prognose
and/or diagnose
severe combined immunodeficiency (SCID).
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent, prognose
and/or diagnose
Wiskott-Aldrich syndrome.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent, prognose
and/or diagnose
X-linked Ig deficiency with hyper IgM.
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In another embodiment, D-SLAM agonists are administered to treat, prevent,
prognose and/or diagnose an autoimmune disease (e.g., rheumatoid arthritis,
systemic lupus
erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic
anemia,
autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, 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, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,
and
autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e.,
Hashimoto's
thyroiditis, 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, 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
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, granulamatous,
degenerative, and
atrophic disorders) or conditions associated with an autoimmune disease. In a
specific
preferred embodiment, rheumatoid arthritis is treated, prevented, prognosed
and/or diagnosed
using D-SLAM and/or other agonists of the invention. In another specific
preferred
embodiment, systemic lupus erythemosus is treated, prevented, prognosed,
and/or diagnosed
using D-SLAM and/or other agonists of the invention. In another specific
preferred
embodiment, idiopathic thrombocytopenia purpura is treated, prevented,
prognosed, and/or
diagnosed using D-SLAM and/or other agonists of the invention. In another
specific preferred
embodiment IgA nephropathy is treated, prevented, prognosed and/or diagnosed
using D-
SLAM and/or other agonists of the invention. In a preferred embodiment, the
autoimmune
diseases and disorders and/or conditions associated with the diseases and
disorders recited
above are treated, prevented, prognosed and/or diagnosed using D-SLAM.
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The invention further provides compositions comprising a D-SLAM
polynucleotide, a
D-SLAM polypeptide, and/or an anti-D-SLAM antibody, for administration to
cells in vitro,
to cells ex vivo, and to cells in vivo, or to a multicellular organism. In
preferred
embodiments, the compositions of the invention comprise a D-SLAM
polynucleotide for
expression of a D-SLAM polypeptide in a host organism for treatment of
disease. In a most
preferred embodiment, the compositions of the invention comprise a D-SLAM
polynucleotide for expression of a D-SLAM polypeptide in a host organism for
treatment of
an immunodeficiency and/or conditions associated with an immunodeficiency.
Particularly
preferred in this regard is expression in a human patient for treatment of a
dysfunction
associated with aberrant endogenous activity of a D-SLAM gene.
The present invention also provides a screening method for identifying
compounds
capable of enhancing or inhibiting a cellular response induced by D-SLAM which
involves
contacting cells which express D-SLAM with the candidate compound, assaying a
cellular
response, and comparing the cellular response to a standard cellular response,
the standard
being assayed when contact is made in absence of the candidate compound;
whereby, an
increased cellular response over the standard indicates that the compound is
an agonist and a
decreased cellular response over the standard indicates that the compound is
an antagonist.
In another embodiment, a method for identifying D-SLAM receptors is provided,
as
well as a screening assay for agonists and antagonists using such receptors.
This assay
involves determining the effect a candidate compound has on D-SLAM binding to
the D-
SLAM receptor. In particular, the method involves contacting a D-SLAM receptor
with a D-
SLAM polypeptide of the invention and a candidate compound and determining
whether D-
SLAM polypeptide binding to the D-SLAM receptor is increased or decreased due
to the
presence of the candidate compound. The agonists may be employed to prevent
septic shock,
inflammation, cerebral malaria, activation of the HIV virus, graft-host
rejection, bone
resorption, rheumatoid arthritis, cachexia (wasting or malnutrition), immune
system function,
lymphoma, and autoimmune disorders (e.g., rheumatoid arthritis and systemic
lupus
erythematosus).
The present inventors have discovered that D-SLAM is highly expressed in
dendritic
cells and lymph node, and to a lesser extent in spleen, thymus, small
intestine, PBLs, bone
marrow, T cell lymphoma, and even lesser extent in placenta and lung. For a
number of
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disorders of these tissues and cells, such as tumor and tumor metastasis,
infection of bacteria,
viruses and other parasites, immunodeficiencies (e.g., chronic variable
immunodeficiency),
septic shock, inflammation, cerebral malaria, activation of the HIV virus,
graft-host rejection,
bone resorption, rheumatoid arthritis, autoimmune diseases (e.g., rheumatoid
arthritis and
5 systemic lupus erythematosus) and cachexia (wasting or malnutrition). It is
believed that
significantly higher or lower levels of D-SLAM gene expression can be detected
in certain
tissues (e.g., bone marrow) or bodily fluids (e.g., serum, plasma, urine,
synovial fluid or
spinal fluid) taken from an individual having such a disorder, relative to a
"standard" D-
SLAM gene expression level, i.e., the D-SLAM expression level in tissue or
bodily fluids
10 from an individual not having the disorder. Thus, the invention provides a
diagnostic method
useful during diagnosis of a disorder, which involves: (a) assaying D-SLAM
gene expression
level in cells or body fluid of an individual; (b) comparing the D-SLAM gene
expression
level with a standard D-SLAM gene expression level, whereby an increase or
decrease in the
assayed D-SLAM gene expression level compared to the standard expression level
is
indicative of a disorder.
An additional embodiment of the invention is related to a method for treating
an
individual in need of an increased or constitutive level of D-SLAM activity in
the body
comprising administering to such an individual a composition comprising a
therapeutically
effective amount of an isolated D-SLAM polypeptide of the invention or an
agonist thereof.
A still further embodiment of the invention is related to a method for
treating an
individual in need of a decreased level of D-SLAM activity in the body
comprising,
administering to such an individual a composition comprising a therapeutically
effective
amount of an D-SLAM antagonist. Preferred antagonists for use in the present
invention are
D-SLAM-specific antibodies.
Brief Description of the Drawings
Figures lA-1D show the nucleotide sequence (SEQ ID NO:1) and the deduced amino
acid sequence (SEQ ID N0:2) of D-SLAM. The predicted leader sequence located
at about
amino acids 1-22 is underlined.
Figure 2 shows the regions of identity between the amino acid sequence of the
D-
SLAM protein and the translation product of the human SLAM (Accession No.
gi/984969)
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(SEQ ID N0:3), determined by BLAST analysis. Identical amino acids between the
two
polypeptides are shaded, while conservative amino acid are boxed. By examining
the regions
of amino acids shaded and/or boxed, the skilled artisan can readily identify
conserved
domains between the two polypeptides. These conserved domains are preferred
embodiments of the present invention.
Figure 3 shows an analysis of the D-SLAM amino acid sequence. Alpha, beta,
turn
and coil regions; hydrophilicity and hydrophobicity; amphipathic regions;
flexible regions;
antigenic index and surface probability are shown, and all were generated
using the default
settings. In the "Antigenic Index or Jameson-Wolf" graph, the positive peaks
indicate
locations of the highly antigenic regions of the D-SLAM protein, i.e., regions
from which
epitope-bearing peptides of the invention can be obtained. The domains defined
by these
graphs are contemplated by the present invention. Tabular representation of
the data
summarized graphically in Figure 3 can be found in Table 1.
The columns are labeled with the headings "Res", "Position", and Roman
Numerals
I-XIV. The column headings refer to the following features of the amino acid
sequence
presented in Figure 3, and Table I: "Res": amino acid residue of SEQ ID N0:2
and Figures
1A and 1B; "Position": position of the corresponding residue within SEQ ID
N0:2 and
Figures 1A and 1B; I: Alpha, Regions - Gamier-Robson; II: Alpha, Regions -
Chou-Fasman;
III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-Fasman; V: Turn,
Regions -
Gamier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions - Gamier-
Robson;
VIII: Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-
Woods; X:
Alpha, Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions -
Eisenberg; XII:
Flexible Regions - Karplus-Schulz; XIII: Antigenic Index - Jameson-Wolf; and
XIV: Surface
Probability Plot - Emini.
Detailed Description
Definitions
The following definitions are provided to facilitate understanding of certain
terms used throughout this specification.
In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is altered "by
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the hand of man" from its natural state. For example, an isolated
polynucleotide could be part
of a vector or a composition of matter, or could be contained within a cell,
and still be
"isolated" because that vector, composition of matter, or particular cell is
not the original
environment of the polynucleotide. Further examples of isolated DNA molecules
include
recombinant DNA molecules maintained in heterologous host cells or purified
(partially or
substantially) DNA molecules in solution. Isolated RNA molecules include in
vivo or in vitro
RNA transcripts of the DNA molecules of the present invention. However, a
nucleic acid
contained in a clone that is a member of a library (e.g., a genomic or cDNA
library) that has
not been isolated from other members of the library (e.g., in the form of a
homogeneous
solution containing the clone and other members of the library) or a
chromosome removed
from a cell or a cell lysate (e.g., a "chromosome spread", as in a karyotype),
or a preparation
of randomly sheared genomic DNA or a preparation of genomic DNA cut with one
or more
restriction enzymes is not "isolated" for the purposes of this invention. As
discussed further
herein, isolated nucleic acid molecules according to the present invention may
be produced
naturally, recombinantly, or synthetically.
In the present invention, a "secreted" D-SLAM protein refers to a protein
capable of being directed to the ER, secretory vesicles, or the extracellular
space as a result of
a signal sequence, as well as a D-SLAM protein released into the extracellular
space without
necessarily containing a signal sequence. If the D-SLAM secreted protein is
released into the
extracellular space, the D-SLAM secreted protein can undergo extracellular
processing to
produce a "mature" D-SLAM protein. Release into the extracellular space can
occur by many
mechanisms, including exocytosis and proteolytic cleavage.
As used herein, a D-SLAM "polynucleotide" refers to a molecule having a
nucleic
acid sequence contained in SEQ ID NO:1 or the cDNA contained within the clone
deposited
with the ATCC. For example, the D-SLAM polynucleotide can contain the
nucleotide
sequence of the full length cDNA sequence, including the 5' and 3'
untranslated sequences,
the coding region, with or without the signal sequence, the secreted protein
coding region, as
well as fragments, epitopes, domains, and variants of the nucleic acid
sequence. Moreover,
as used herein, a D-SLAM "polypeptide" refers to a molecule having the
translated amino
acid sequence generated from the polynucleotide as broadly defined.
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13
In specific embodiments, the polynucleotides of the invention are less than
300 kb,
200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length. In a further
embodiment,
polynucleotides of the invention comprise at least 15 contiguous nucleotides
of D-SLAM
coding sequence, but do not comprise all or a portion of any D-SLAM intron. In
another
embodiment, the nucleic acid comprising D-SLAM coding sequence does not
contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the D-SLAM gene in the
genome).
In the present invention, the full length D-SLAM sequence identified as SEQ ID
NO:1 was generated by overlapping sequences of the deposited clone (contig
analysis). A
representative clone containing all or most of the sequence for SEQ ID NO:1
was deposited.
with the American Type Culture Collection ("ATCC") on February 6, 1998, and
was given
the ATCC Deposit Number 209623. 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.
A D-SLAM "polynucleotide" also includes those polynucleotides capable of
hybridizing, under stringent hybridization conditions, to sequences contained
in SEQ ID
NO:1, the complement thereof, or the cDNA within the deposited clone.
"Stringent
hybridization conditions" refers to an overnight incubation at 42~C in a
solution comprising
50% formamide, 5x SSC (750 mM NaCI, 75 mM sodium citrate), 50 mM sodium
phosphate
(pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured,
sheared
salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65~C.
Also contemplated are nucleic acid molecules that hybridize to the D-SLAM
polynucleotides at moderatetly high stringency hybridization conditions.
Changes in the
stringency of hybridization and signal detection are primarily accomplished
through the
manipulation of formamide concentration (lower percentages of formamide result
in lowered
stringency); salt conditions, or temperature. For example, moderately high
stringency
conditions include an overnight incubation at 37~C in a solution comprising 6X
SSPE (20X
SSPE = 3M NaCI; 0.2M NaHZPO~; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide,
100
ug/ml salmon sperm blocking DNA; followed by washes at 50~C with 1XSSPE, 0.1%
SDS.
In addition, to achieve even lower stringency, washes performed following
stringent
hybridization can be done at higher salt concentrations (e.g. 5X SSC).
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14
Note that variations in the above conditions may be accomplished through the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in
hybridization experiments. Typical blocking reagents include Denhardt's
reagent, BLOTTO,
heparin, denatured salmon sperm DNA, and commercially available proprietary
formulations.
The inclusion of specific blocking reagents may require modification of the
hybridization
conditions described above, due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as
any
3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a
complementary
stretch of T (or U) residues, would not be included in the definition of
"polynucleotide," since
such a polynucleotide would hybridize to any nucleic acid molecule containing
a poly (A)
stretch or the complement thereof (e.g., practically any double-stranded cDNA
clone).
The D-SLAM polynucleotide can be composed of any polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or
DNA.
For example, D-SLAM polynucleotides can be composed of single- and double-
stranded
DNA, DNA that is a mixture of single- and double-stranded regions, single- and
double-
stranded RNA, and RNA that is mixture of single- and double-stranded regions,
hybrid
molecules comprising DNA and RNA that may be single-stranded or, more
typically, double-
stranded or a mixture of single- and double-stranded regions. In addition, the
D-SLAM
polynucleotides can be composed of triple-stranded regions comprising RNA or
DNA or both
RNA and DNA. D-SLAM polynucleotides may also contain one or more modified
bases or
DNA or RNA backbones modified for stability or for other reasons. "Modified"
bases
include, for example, tritylated bases and unusual bases such as inosine. A
variety of
modifications can be made to DNA and RNA; thus, "polynucleotide" embraces
chemically,
enzymatically, or metabolically modified forms.
D-SLAM polypeptides can be composed of amino acids joined to each other by
peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may
contain amino acids
other than the 20 gene-encoded amino acids. The D-SLAM polypeptides may be
modified by
either natural processes, such as posttranslational processing, or by chemical
modification
techniques which are well known in the art. Such modifications are well
described in basic
texts and in more detailed monographs, as well as in a voluminous research
literature.
Modifications can occur anywhere in the D-SLAM polypeptide, including the
peptide
CA 02382659 2002-O1-29
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backbone, the amino acid side-chains and the amino or carboxyl termini. It
will be
appreciated that the same type of modification may be present in the same or
varying degrees
at several sites in a given D-SLAM polypeptide. Also, a given D-SLAM
polypeptide may
contain many types of modifications. D-SLAM polypeptides may be branched, for
example,
5 as a result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic,
branched, and branched cyclic.D-SLAM polypeptides may result from
posttranslation natural
processes or may be made by synthetic methods. Modifications include, but are
not limited
to, acetylation, acylation, ADP-ribosylation, amidation, biotinylation,
covalent attachment of
flavin, covalent attachment of a heme moiety, covalent attachment of a
nucleotide or
10 nucleotide derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment
of phosphotidylinositol, cross-linking, cyclization, derivatization by known
protecting/blocking groups, disulfide bond formation, demethylation, formation
of covalent
cross-links, formation of cysteine, formation of pyroglutamate, formylation,
gamma-
carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination,
linkage to an
15 antibody molecule or other cellular ligand, methylation, myristoylation,
oxidation,
pegylation, proteolytic processing (e.g., cleavage), phosphorylation,
prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated addition of
amino acids to
proteins such as arginylation, and ubiquitination. (See, for instance,
PROTEINS -
STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-
12 ( 1983); Seifter et al., Meth Enzymol 182:626-646 ( 1990); Rattan et al.,
Ann NY Acad Sci
663:48-62 (1992).) 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.
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
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16
polypeptides may also be modified with a detectable label, such as an
enzymatic, fluorescent,
isotopic or affinity label to allow for detection and isolation of the
protein. In addition,
polypeptides of the invention may be modified by iodination.
In one embodiment, D-SLAM polypeptides of the invention may also be labeled
with
biotin. In other related embodiments, biotinylated D-SLAM polypeptides of the
invention
may be used, for example, as an imaging agent or as a means of identifying one
or more D
SLAM receptors) or other coreceptor or coligand molecules.
Also provided by the invention are chemically modified derivatives of D-SLAM
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, two, three or more attached chemical moieties.
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 kDa.
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
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17
al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides
18:2745-2750 ( 1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (
1999), the
disclosures of each of which are incorporated herein by reference.
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 protein.
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 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.
As suggested above, polyethylene glycol may be attached to proteins 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 protein or to more than one type of amino acid residue (e.g., lysine,
histidine, aspartic
acid, glutamic acid, cysteine and combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the N-terminus.
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
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18
N-terminally pegylated material from a population of pegylated protein
molecules. Selective
proteins chemically modified at the N-terminus modification 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 protein.
Under the appropriate reaction conditions, substantially selective
derivatization of the protein
at the N-terminus with a carbonyl group containing polymer is achieved.
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. Thera. Drug
Carrier Sys. 9:249-
304 (1992); Francis et al., Intern. J. of Hematol. 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.
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
(CISOzCH,CF3). Upon reaction of protein with tresylated MPEG, polyethylene
glycol is
directly attached to amine groups of the protein. Thus, the invention includes
protein
polyethylene glycol conjugates produced by reacting proteins of the invention
with a
polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins 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 protein by a linker can also be produced by .reaction of
proteins 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
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19
by reference. Pegylated protein products produced using the reaction
chemistries set out
herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each protein of the
invention
(i.e., the degree of substitution) may also vary. For example, the pegylated
proteins of the
invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
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 protein molecule.
Methods for
determining the degree of substitution are discussed, for example, in Delgado
et al., Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 ( 1992).
The D-SLAM polypeptides can be recovered and purified by known methods which
include, but are not limited to, ammonium sulfate or ethanol precipitation,
acid extraction,
anion or canon exchange chromatography, phosphocellulose chromatography,
hydrophobic
interaction chromatography, affinity chromatography, hydroxylapatite
chromatography and
lectin chromatography. Most preferably, high performance liquid chromatography
("HPLC")
is employed for purification.
"SEQ ID NO:1 " refers to a D-SLAM polynucleotide sequence while "SEQ ID N0:2"
refers to a D-SLAM polypeptide sequence.
A D-SLAM polypeptide "having biological activity" refers to polypeptides
exhibiting
activity similar, but not necessarily identical to, an activity of a D-SLAM
polypeptide,
including mature forms, as measured in a particular biological assay, with or
without dose
dependency. In the case where dose dependency does exist, it need not be
identical to that of
the D-SLAM polypeptide, but rather substantially similar to the dose-
dependence in a given
activity as compared to the D-SLAM polypeptide (i.e., the candidate
polypeptide will exhibit
greater activity or not more than about 25-fold less and, preferably, not more
than about
tenfold less activity, and most preferably, not more than about three-fold
less activity relative
to the D-SLAM polypeptide.)
D-SLAM Polynucleotides and Polypentides
Clone HDPJ039 was isolated from a dendritic cell cDNA library. This clone
contains
the entire coding region identified as SEQ ID N0:2. The deposited clone
contains a cDNA
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having a total of 3220 nucleotides, which encodes a predicted open reading
frame of 285
amino acid residues. (See Figures lA-1D.) The open reading frame begins at a N-
terminal
methionine located at nucleotide position 92, and ends at a stop codon at
nucleotide position
947. The predicted molecular weight of the D-SLAM protein should be about 34.2
kDa.
5 Subsequent Northern analysis also showed D-SLAM expression in dendritic
cells, T
cell lymphoma, lymph node, spleen, thymus, small intestine, and uterus
tissues, a pattern
consistent with hematopoietic specific expression. Expression is highest in
tissues involved
in immune recognition, consistent with the enriched expression in dendritic
cells and APC's.
A single primary transcript of approximately 3.5-4.0 kb is observed, with a
minor transcript
10 of 7-9 kb that likely represents an unprocessed RNA precursor. The
expression of the major
3.5-4 kb transcript is highest in lymph node, spleen, thymus, and, to a lesser
degree, in small
intestine. The highest expression of the 7-9 kb transcript is observed in the
uterus.
Using BLAST analysis, SEQ ID N0:2 was found to be homologous to members of
the Secreted Lymphocyte Activation Molecule (SLAM) family. Particularly, SEQ
ID N0:2
15 contains domains homologous to the translation product of the human mRNA
for SLAM
(Accession No. gi/984969) (Figure 2) (SEQ ID N0:3), including the following
conserved
domains: (a) a predicted transmembrane domain located at about amino acids 233-
255; (b) a
predicted extracellular domain located at about amino acids 23-232; and (c) a
predicted
intracellular domain located at about amino acids 256-285. These polypeptide
fragments of
20 D-SLAM are specifically contemplated in the present invention. Because SLAM
(Accession
No. gi/984969) is thought to be important in the activation and proliferation
of T- and B-
cells, the homology between SLAM (Accession No. gi/984969) and D-SLAM suggests
that
D-SLAM may also be involved in the activation and proliferation of T- and B-
cells.
Moreover, the encoded polypeptide has a predicted leader sequence located at
about
amino acids 1-22. (See Figures lA-1D.) Also shown in Figures lA-1D, the
predicted
secreted form of D-SLAM encompasses about amino acids 23-232. These
polypeptide
fragments of D-SLAM are specifically contemplated in the present invention.
The D-SLAM nucleotide sequence identified as SEQ ID NO:1 was assembled from
partially homologous ("overlapping") sequences obtained from the deposited
clone, and in
some cases, from additional related DNA clones. The overlapping sequences were
assembled
into a single contiguous sequence of high redundancy (usually three to five
overlapping
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21
sequences at each nucleotide position), resulting in a final sequence
identified as SEQ ID
NO:1.
Therefore, SEQ ID NO:1 and the translated SEQ ID N0:2 are sufficiently
accurate
and otherwise suitable for a variety of uses well known in the art and
described further below.
For instance, SEQ ID NO:1 is useful for designing nucleic acid hybridization
probes that will
detect nucleic acid sequences contained in SEQ ID NO:1 or the cDNA contained
in the
deposited clone. These probes will also hybridize to nucleic acid molecules in
biological
samples, thereby enabling a variety of forensic and diagnostic methods of the
invention.
Similarly, polypeptides identified from SEQ ID N0:2 may be used to generate
antibodies
which bind specifically to D-SLAM .
Nevertheless, DNA sequences generated by sequencing reactions can contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or deletions
of nucleotides in the generated DNA sequence. The erroneously inserted or
deleted
nucleotides cause frame shifts in the reading frames of the predicted amino
acid sequence. In
these cases, the predicted amino acid sequence diverges from the actual amino
acid sequence,
even though the generated DNA sequence may be greater than 99.9% identical to
the actual
DNA sequence (for example, one base insertion or deletion in an open reading
frame of over
1000 bases).
Accordingly, for those applications requiring precision in the nucleotide
sequence or
the amino acid sequence, the present invention provides not only the generated
nucleotide
sequence identified as SEQ ID NO:1 and the predicted translated amino acid
sequence
identified as SEQ ID N0:2, but also a sample of plasmid DNA containing a human
cDNA of
D-SLAM deposited with the ATCC. The nucleotide sequence of the deposited D-
SLAM
clone can readily be determined by sequencing the deposited clone in
accordance with known
methods. The predicted D-SLAM amino acid sequence can then be verified from
such
deposits. Moreover, the amino acid sequence of the protein encoded by the
deposited clone
can also be directly determined by peptide sequencing or by expressing the
protein in a
suitable host cell containing the deposited human D-SLAM cDNA, collecting the
protein, and
determining its sequence.
The present invention also relates to the D-SLAM gene corresponding to SEQ ID
NO:1, SEQ ID N0:2, or the deposited clone. The D-SLAM gene can be isolated in
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22
accordance with known methods using the sequence information disclosed herein.
Such
methods include preparing probes or primers from the disclosed sequence and
identifying or
amplifying the D-SLAM gene from appropriate sources of genomic material.
Also provided in the present invention are species homologs of D-SLAM. Species
homologs may be isolated and identified by making suitable probes or primers
from the
sequences provided herein and screening a suitable nucleic acid source for the
desired
homologue.
The D-SLAM polypeptides can be prepared in any suitable manner. Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood in
the art.
The D-SLAM polypeptides may be in the form of the secreted protein, including
the
mature form, or may be a part of a larger protein, such as a fusion protein
(see below). It is
often advantageous to include an additional amino acid sequence which contains
secretory or
leader sequences, pro-sequences, sequences which aid in purification, such as
multiple
histidine residues, or an additional sequence for stability during recombinant
production.
D-SLAM polypeptides are preferably provided in an isolated form, and
preferably are
substantially purified. A recombinantly produced version of a D-SLAM
polypeptide,
including the secreted polypeptide, can be substantially purified by the one-
step method
described in Smith and Johnson, Gene 67:31-40 ( 1988). D-SLAM polypeptides
also can be
purified from natural or recombinant sources using antibodies of the invention
raised against
the D-SLAM protein in methods which are well known in the art.
Polynucleotide and Poly~entide Variants
"Variant" refers to a polynucleotide or polypeptide differing from the D-SLAM
polynucleotide or polypeptide, but retaining essential properties thereof.
Generally, variants
are overall closely similar, and, in many regions, identical to the D-SLAM
polynucleotide or
polypeptide.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended that the
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23
nucleotide sequence of the polynucleotide is identical to the reference
sequence except that
the polynucleotide sequence may include up to five point mutations per each
100 nucleotides
of the reference nucleotide sequence encoding the D-SLAM polypeptide. In other
words, to
obtain a polynucleotide having a nucleotide sequence at least 95% identical to
a reference
nucleotide sequence, up to 5% of the nucleotides in the reference sequence may
be deleted or
substituted with another nucleotide, or a number of nucleotides up to 5% of
the total
nucleotides in the reference sequence may be inserted into the reference
sequence. The query
sequence may be an entire sequence shown of SEQ ID NO:I, the ORF (open reading
frame),
or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is
at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a
nucleotide
sequence of the presence invention can be determined conventionally using
known computer
programs. A preferred method for determining the best overall match between a
query
sequence (a sequence of the present invention) and a subject sequence, also
referred to as a
global sequence alignment, can be determined using the FASTDB computer program
based
on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245.) In a
sequence
alignment the query and subject sequences are both DNA sequences. An RNA
sequence can
be compared by converting U's to T's. The result of said global sequence
alignment is in
percent identity. Preferred parameters used in a FASTDB alignment of DNA
sequences to
calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining
Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap
Size
Penalty 0.05, Window Size=500 or the length of the subject nucleotide
sequence, whichever
is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3'
deletions, not because of internal deletions, a manual correction must be made
to the results.
This is because the FASTDB program does not account for 5' and 3' truncations
of the
subject sequence when calculating percent identity. For subject sequences
truncated at the 5'
or 3' ends, relative to the query sequence, the percent identity is corrected
by calculating the
number of bases of the query sequence that are 5' and 3' of the subject
sequence, which are
not matched/aligned, as a percent of the total bases of the query sequence.
Whether a
nucleotide is matched/aligned is determined by results of the FASTDB sequence
alignment.
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24
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 corrected score is what is used for the purposes of the present
invention. Only bases
outside the 5' and 3' bases of the subject sequence, as displayed by the
FASTDB alignment,
which are not matched/aligned with the query sequence, are calculated for the
purposes of
manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query
sequence to
determine percent identity. The deletions occur at the 5' end of the subject
sequence and
therefore, the FASTDB alignment does not show a matched/alignment of the first
10 bases at
5' end. The 10 unpaired bases represent 10% of the sequence (number of bases
at the 5' and
3' ends not matched/total number of bases in the query sequence) so 10% is
subtracted from
the percent identity score calculated by the FASTDB program. If the remaining
90 bases
were perfectly matched the final percent identity would be 90%. In another
example, a 90
base subject sequence is compared with a 100 base query sequence. This time
the deletions
are internal deletions so that there are no bases on the 5' or 3' 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 bases 5' and 3' of the
subject sequence
which are not matched/aligned with the query sequence are manually corrected
for. No other
manual corrections are to made for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that the
amino acid sequence of the subject polypeptide is identical to the query
sequence except that
the subject polypeptide sequence may include up to five amino acid alterations
per each 100
amino acids of the query amino acid sequence. In other words, to obtain a
polypeptide
having an amino acid sequence at least 95% identical to a query amino acid
sequence, up to
5% of the amino acid residues in the subject sequence may be inserted,
deleted, (indels) or
substituted with another amino acid. 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.
As a practical matter, whether any particular polypeptide is at least 80%,
85%, 90%,
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92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid
sequences
shown in SEQ ID N0:2 or to the amino acid sequence encoded by deposited DNA
clone can
be determined conventionally using known computer programs. A preferred method
for
determining the best overall match between a query sequence (a sequence of the
present
5 invention) and a subject sequence, also referred to as a global sequence
alignment, can be
determined using the FASTDB computer program based on the algorithm of Brutlag
et al.
(Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and
subject
sequences are either both nucleotide sequences or both amino acid sequences.
The result of
said global sequence alignment is in percent identity. Preferred parameters
used in a
10 FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=l,
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.
If the subject sequence is shorter than the query sequence due to N- or C-
terminal
15 deletions, not because of internal deletions, a manual correction must be
made to the results.
This is because 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-
20 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. 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
25 percent identity score is what is used for the purposes of the present
invention. 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
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26
subject sequence and therefore, the FASTDB alignment does not show a
matching/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 to
made for the
purposes of the present invention.
The D-SLAM variants may contain alterations in the coding regions, non-coding
regions, or both. Especially preferred are polynucleotide variants containing
alterations
which produce silent substitutions, additions, or deletions, but do not alter
the properties or
activities of the encoded polypeptide. Nucleotide variants produced by silent
substitutions
due to the degeneracy of the genetic code are preferred. Moreover, variants in
which 5-10, 1-
5, or 1-2 amino acids are substituted, deleted, or added in any combination
are also preferred.
D-SLAM polynucleotide variants can be produced for a variety of reasons, e.g.,
to optimize
codon expression for a particular host (change codons in the human mRNA to
those preferred
by a bacterial host such as E. coli).
Naturally occurring D-SLAM variants are called "allelic variants," and refer
to one of
several alternate forms of a gene occupying a given locus on a chromosome of
an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New York ( 1985).) These allelic
variants can
vary at either the polynucleotide and/or polypeptide level. Alternatively, non-
naturally
occurring variants may be produced by mutagenesis techniques or by direct
synthesis.
Using known methods of protein engineering and recombinant DNA technology,
variants may be generated to improve or alter the characteristics of the D-
SLAM
polypeptides. For instance, one or more amino acids can be deleted from the N-
terminus or
C-terminus of the secreted protein without substantial loss of biological
function. The
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27
authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant
KGF proteins
having heparin binding activity even after deleting 3, 8, or 27 amino-terminal
amino acid
residues. Similarly, Interferon gamma exhibited up to ten times higher
activity after deleting
8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et
al., J.
Biotechnology 7:199-216 ( 1988).)
Moreover, ample evidence demonstrates that variants often retain a biological
activity
similar to that of the naturally occurring protein. For example, Gayle and
coworkers (J. Biol.
Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human
cytokine
IL-la. They used random mutagenesis to generate over 3,500 individual IL-la
mutants that
averaged 2.5 amino acid changes per variant over the entire length of the
molecule. Multiple
mutations were examined at every possible amino acid position. The
investigators found that
"[m]ost of the molecule could be altered with little effect on either [binding
or biological
activity]." (See, Abstract.) In fact, only 23 unique amino acid sequences, out
of more than
3,500 nucleotide sequences examined, produced a protein that significantly
differed in
activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-
terminus of a polypeptide results in modification or loss of one or more
biological functions,
other biological activities may still be retained. For example, the ability of
a deletion variant
to induce and/or to bind antibodies which recognize the secreted form will
likely be retained
when less than the majority of the residues of the secreted form are removed
from the N-
terminus or C-terminus. Whether a particular polypeptide lacking N- or C-
terminal residues
of a protein retains such immunogenic activities can readily be determined by
routine
methods described herein and otherwise known in the art.
Thus, the invention further includes D-SLAM polypeptide variants which show
substantial biological activity. Such variants include deletions, insertions,
inversions,
repeats, and substitutions selected according to general rules known in the
art so as have little
effect on activity.
The present application is directed to nucleic acid molecules at least 80%,
85%, 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences
disclosed herein,
(e.g., encoding a polypeptide having the amino acid sequence of an N and/or C
terminal
deletion disclosed below as m-n of SEQ ID N0:2), irrespective of whether they
encode a
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28
polypeptide having D-SLAM functional activity. This is because even where a
particular
nucleic acid molecule does not encode a polypeptide having D-SLAM functional
activity, one
of skill in the art would still know how to use the nucleic acid molecule, for
instance, as a
hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the
nucleic acid
molecules of the present invention that do not encode a polypeptide having D-
SLAM
functional activity include, inter alia, ( 1 ) isolating a D-SLAM gene or
allelic or splice
variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH")
to metaphase
chromosomal spreads to provide precise chromosomal location of the D-SLAM
gene, as
described in Verma et al., Human Chromosomes: A Manual of Basic Techniques,
Pergamon
Press, New York (1988); and (3) Northern Blot analysis for detecting D-SLAM
mRNA
expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 80%,
85%,
90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences
disclosed
herein, which do, in fact, encode a polypeptide having D-SLAM functional
activity. By "a
polypeptide having D-SLAM functional activity" is intended polypeptides
exhibiting activity
similar, but not necessarily identical, to a functional activity of the D-SLAM
polypeptides of
the present invention (e.g., complete (full-length) D-SLAM, mature D-SLAM and
soluble D-
SLAM (e.g., having sequences contained in the extracellular domain of D-SLAM)
as
measured, for example, in a particular immunoassay or biological assay. For
example, a D-
SLAM functional activity can routinely be measured by determining the ability
of a D-SLAM
polypeptide to bind a D-SLAM ligand. D-SLAM functional activity may also be
measured by
determining the ability of a polypeptide, such as cognate ligand which is free
or expressed on
a cell surface, to induce cells expressing the polypeptide.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in
the art
will immediately recognize that a large number of the nucleic acid molecules
having a
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to
the
nucleic acid sequence of the deposited cDNA, the nucleic acid sequence shown
in Figure 1
(SEQ ID NO:1), or fragments thereof, will encode polypeptides "having D-SLAM
functional
activity." In fact, since degenerate variants of any of these nucleotide
sequences all encode
the same polypeptide, in many instances, this will be clear to the skilled
artisan even without
performing the above described comparison assay. It will be further recognized
in the art
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29
that, for such nucleic acid molecules that are not degenerate variants, a
reasonable number
will also encode a polypeptide having D-SLAM functional activity. This is
because the
skilled artisan is fully aware of amino acid substitutions that are either
less likely or not likely
to significantly effect protein function (e.g., replacing one aliphatic amino
acid with a second
aliphatic amino acid), as further described below.
For example, guidance concerning how to make phenotypically silent amino acid
substitutions is provided in Bowie, J. U. et al., Science 247:1306-1310
(1990), wherein the
authors indicate that there are two main strategies for studying the tolerance
of an amino acid
sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by
natural
selection during the process of evolution. By comparing amino acid sequences
in different
species, conserved amino acids can be identified. These conserved amino acids
are likely
important for protein function. In contrast, the amino acid positions where
substitutions have
been tolerated by natural selection indicates that these positions are not
critical for protein
function. Thus, positions tolerating amino acid substitution could be modified
while still
maintaining biological activity of the protein.
The second strategy uses genetic engineering to introduce amino acid changes
at
specific positions of a cloned gene to identify regions critical for protein
function. For
example, site directed mutagenesis or alanine-scanning mutagenesis
(introduction of single
alanine mutations at every residue in the molecule) can be used. (Cunningham
and Wells,
Science 244:1081-1085 (1989).) The resulting mutant molecules can then be
tested for
biological activity.
As the authors state, these two strategies 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 certain amino acid positions in the protein.
For example, most
buried (within the tertiary structure of the protein) amino acid residues
require nonpolar side
chains, whereas few features of surface side chains are generally conserved.
Moreover,
tolerated conservative amino acid substitutions involve replacement of the
aliphatic or
hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl
residues Ser and
Thr; replacement of the acidic residues Asp and Glu; replacement of the amide
residues Asn
and Gln, replacement of the basic residues Lys, Arg, and His; replacement of
the aromatic
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residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids
Ala, Ser, Thr,
Met, and Gly.
For example, site directed changes at the amino acid level of D-SLAM can be
made
by replacing a particular amino acid with a conservative amino acid. Preferred
conservative
5 mutations include: Ml replaced with A, G, I, L, S, T, or V; V2 replaced with
A, G, I, L, S, T,
or M; M3 replaced with A, G, I, L, S, T, or V; R4 replaced with H, or K; L6
replaced with A,
G, I, S, T, M, or V; W7 replaced with F, or Y; S8 replaced with A, G, I, L, T,
M, or V; L9
replaced with A, G, I, S, T, M, or V; L10 replaced with A, G, I, S, T, M, or
V; L11 replaced
with A, G, I, S, T, M, or V; W 12 replaced with F, or Y; E 13 replaced with D;
A 14 replaced
10 with G, I, L, S, T, M, or V; L15 replaced with A, G, I, S, T, M, or V; L16
replaced with A, G,
I, S, T, M, or V; I18 replaced with A, G, L, S, T, M, or V; T19 replaced with
A, G, I, L, S, M,
or V; V20 replaced with A, G, I, L, S, T, or M; T21 replaced with A, G, I, L,
S, M, or V; G22
replaced with A, I, L, S, T, M, or V; A23 replaced with G, I, L, S, T, M, or
V; Q24 replaced
with N; V25 replaced with A, G, I, L, S, T, or M; L26 replaced with A, G, I,
S, T, M, or V;
15 S27 replaced with A, G, I, L, T, M, or V; K28 replaced with H, or R; V29
replaced with A,
G, I, L, S, T, or M; G30 replaced with A, I, L, S, T, M, or V; G31 replaced
with A, I, L, S, T,
M, or V; S32 replaced with A, G, I, L, T, M, or V; V33 replaced with A, G, I,
L, S, T, or M;
L34 replaced with A, G, I, S, T, M, or V; L35 replaced with A, G, I, S, T, M,
or V; V36
replaced with A, G, I, L, S, T, or M; A37 replaced with G, I, L, S, T, M, or
V; A38 replaced
20 with G, I, L, S, T, M, or V; R39 replaced with H, or K; G42 replaced with
A, I, L, S, T, M, or
V; F43 replaced with W, or Y; Q44 replaced with N; V45 replaced with A, G, I,
L, S, T, or
M; R46 replaced with H, or K; E47 replaced with D; A48 replaced with G, I, L,
S, T, M, or
V; I49 replaced with A, G, L, S, T, M, or V; W50 replaced with F, or Y; R51
replaced with
H, or K; S52 replaced with A, G, I, L, T, M, or V; L53 replaced with A, G, I,
S, T, M, or V;
25 W54 replaced with F, or Y; S56 replaced with A, G, I, L, T, M, or V; E57
replaced with D;
E58 replaced with D; L59 replaced with A, G, I, S, T, M, or V; L60 replaced
with A, G, I, S,
T, M, or V; A61 replaced with G, I, L, S, T, M, or V; T62 replaced with A, G,
I, L, S, M, or
V; F63 replaced with W, or Y; F64 replaced with W, or Y; R65 replaced with H,
or K; G66
replaced with A, I, L, S, T, M, or V; S67 replaced with A, G, I, L, T, M, or
V; L68 replaced
30 with A, G, I, S, T, M, or V; E69 replaced with D; T70 replaced with A, G,
I, L, S, M, or V;
L71 replaced with A, G, I, S, T, M, or V; Y72 replaced with F, or W; H73
replaced with K, or
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31
R; S74 replaced with A, G, I, L, T, M, or V; R75 replaced with H, or K; F76
replaced with
W, or Y; L77 replaced with A, G, I, S, T, M, or V; G78 replaced with A, I, L,
S, T, M, or V;
R79 replaced with H, or K; A80 replaced with G, I, L, S, T, M, or V; Q81
replaced with N;
L82 replaced with A, G, I, S, T, M, or V; H83 replaced with K, or R; S84
replaced with A, G,
I, L, T, M, or V; N85 replaced with Q; L86 replaced with A, G, I, S, T, M, or
V; S87 replaced
with A, G, I, L, T, M, or V; L88 replaced with A, G, I, S, T, M, or V; E89
replaced with D;
L90 replaced with A, G, I, S, T, M, or V; G91 replaced with A, I, L, S, T, M,
or V; L93
replaced with A, G, I, S, T, M, or V; E94 replaced with D; S95 replaced with
A, G, I, L, T,
M, or V; G96 replaced with A, I, L, S, T, M, or V; D97 replaced with E; S98
replaced with A,
G, I, L, T, M, or V; G99 replaced with A, I, L, S, T, M, or V; N100 replaced
with Q; F101
replaced with W, or Y; S 102 replaced with A, G, I, L, T, M, or V; V 103
replaced with A, G,
I, L, S, T, or M; L104 replaced with A, G, I, S, T, M, or V; M105 replaced
with A, G, I, L, S,
T, or V; V106 replaced with A, G, I, L, S, T, or M; D107 replaced with E; T108
replaced
with A, G, I, L, S, M, or V; 8109 replaced with H, or K; 6110 replaced with A,
I, L, S, T, M,
or V; Q 111 replaced with N; W 113 replaced with F, or Y; T 114 replaced with
A, G, I, L, S,
M, or V; Q115 replaced with N; T116 replaced with A, G, I, L, S, M, or V; L117
replaced
with A, G, I, S, T, M, or V; Q118 replaced with N; Ll 19 replaced with A, G,
I, S, T, M, or V;
K120 replaced with H, or R; V121 replaced with A, G, I, L, S, T, or M; Y122
replaced with
F, or W; D123 replaced with E; A124 replaced with G, I, L, S, T, M, or V; V125
replaced
with A, G, I, L, S, T, or M; R 127 replaced with H, or K; V 129 replaced with
A, G, I, L, S, T,
or M; V 130 replaced with A, G, I, L, S,
T, or M; Q 131 replaced with N; V 132 replaced with A, G, I, L, S, T, or M;
F133 replaced
with W, or Y; I134 replaced with A, G, L, S, T, M, or V; A135 replaced with G,
I, L, S, T, M,
or V; V136 replaced with A, G, I, L, S, T, or M; E137 replaced with D; 8138
replaced with
H, or K; D 139 replaced with E; A 140 replaced with G, I, L, S, T, M, or V; Q
141 replaced
with N; S143 replaced with A, G, I, L, T, M, or V; K144 replaced with H, or R;
T145
replaced with A, G, I, L, S, M, or V; Q147 replaced with N; V148 replaced with
A, G, I, L, S,
T, or M; F149 replaced with W, or Y; L150 replaced with A, G, I, S, T, M, or
V; S151
replaced with A, G, I, L, T, M, or V; W153 replaced with F, or Y; A154
replaced with G, I,
L, S, T, M, or V; N156 replaced with Q; I157 replaced with A, G, L, S, T, M,
or V; S158
replaced with A, G, I, L, T, M, or V; E159 replaced with D; I160 replaced with
A, G, L, S, T,
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32
M, or V; T161 replaced with A, G, I, L, S, M, or V; Y162 replaced with F, or
W; S163
replaced with A, G, I, L, T, M, or V; W 164 replaced with F, or Y; 8165
replaced with H, or
K; R 166 replaced with H, or K; E 167 replaced with D; T 168 replaced with A,
G, I, L, S, M,
or V; T 169 replaced with A, G, I, L, S, M, or V; M 170 replaced with A, G, I,
L, S, T, or V;
D 171 replaced with E; F 172 replaced with W, or Y; G 173 replaced with A, I,
L, S, T, M, or
V; M174 replaced with A, G, I, L, S, T, or V; E175 replaced with D; H177
replaced with K,
or R; 5178 replaced with A, G, I, L, T, M, or V; L179 replaced with A, G, I,
S, T, M, or V;
F 180 replaced with W, or Y; T 181 replaced with A, G, I, L, S, M, or V; D 182
replaced with
E; 6183 replaced with A, I, L, S, T, M, or V; Q184 replaced with N; V185
replaced with A,
G, I, L, S, T, or M; L186 replaced with A, G, I, S, T, M, or V; 5187 replaced
with A, G, I, L,
T, M, or V; I188 replaced with A, G, L, S, T, M, or V; S 189 replaced with A,
G, I, L, T, M,
or V; L190 replaced with A, G, I, S, T, M, or V; 6191 replaced with A, I, L,
S, T, M, or V;
6193 replaced with A, I, L, S, T, M, or V; D194 replaced with E; 8195 replaced
with H, or
K; D196 replaced with E; V197 replaced with A, G, I, L, S, T, or M; A198
replaced with G,
I, L, S, T, M, or V; Y199 replaced with F, or W; S200 replaced with A, G, I,
L, T, M, or V;
I202 replaced with A, G, L, S, T, M, or V; V203 replaced with A, G, I, L, S,
T, or M; S204
replaced with A, G, I, L, T, M, or V; N205 replaced with Q; V207 replaced with
A, G, I, L, S,
T, or M; S208 replaced with A, G, I, L, T, M, or V; W209 replaced with F, or
Y; D210
replaced with E; L211 replaced with A, G, I, S, T, M, or V; A212 replaced with
G, I, L, S, T,
M, or V; T213 replaced with A, G, I, L, S, M, or V; V214 replaced with A, G,
I, L, S, T, or
M; T215 replaced with A, G, I, L, S, M, or V; W217 replaced with F, or Y; D218
replaced
with E; S219 replaced with A, G, I, L, T, M, or V; H221 replaced with K, or R;
H222
replaced with K, or R; E223 replaced with D; A224 replaced with G, I, L, S, T,
M, or V;
A225 replaced with G, I, L, S, T, M, or V; 6227 replaced with A, I, L, S, T,
M, or V; K228
replaced with H, or R; A229 replaced with G, I, L, S, T, M, or V; 5230
replaced with A, G, I,
L, T, M, or V; Y231 replaced with F, or W; K232 replaced with H, or R; D233
replaced with
E; V234 replaced with A, G, I, L, S, T, or M; L235 replaced with A, G, I, S,
T, M, or V;
L236 replaced with A, G, I, S, T, M, or V; V237 replaced with A, G, I, L, S,
T, or M; V238
replaced with A, G, I, L, S, T, or M; V239 replaced with A, G, I, L, S, T, or
M; V241
replaced with A, G, I, L, S, T, or M; 5242 replaced with A, G, I, L, T, M, or
V; L243
CA 02382659 2002-O1-29
WO 01/11046 PCT/US00/21130
33
replaced with A, G, I, S, T, M, or V; L244 replaced with A, G, I, S, T, M, or
V; L245
replaced with A, G, I, S, T, M, or V; M246 replaced with A, G, I, L, S, T, or
V; L247
replaced with A, G, I, S, T, M, or V; V248 replaced with A, G, I, L, S, T, or
M; T249
replaced with A, G, I, L, S, M, or V; L250 replaced with A, G, I, S, T, M, or
V; F251
replaced with W, or Y; S252 replaced with A, G, I, L, T, M, or V; A253
replaced with G, I,
L, S, T, M, or V; W254 replaced with F, or Y; H255 replaced with K, or R; W256
replaced
with F, or Y; S260 replaced with A, G, I, L, T, M, or V; 6261 replaced with A,
I, L, S, T, M,
or V; K262 replaced with H, or R; K263 replaced with H, or R; K264 replaced
with H, or R;
K265 replaced with H, or R; D266 replaced with E; V267 replaced with A, G, I,
L, S, T, or
M; H268 replaced with K, or R; A269 replaced with G, I, L, S, T, M, or V; D270
replaced
with E; 8271 replaced with H, or K; V272 replaced with A, G, I, L, S, T, or M;
6273
replaced with A, I, L, S, T, M, or V; E275 replaced with D; T276 replaced with
A, G, I, L, S,
M, or V; E277 replaced with D; N278 replaced with Q; L280 replaced with A, G,
I, S, T, M,
or V; V281 replaced with A, G, I, L, S, T, or M; Q282 replaced with N; D283
replaced with
E; L284 replaced with A, G, I, S, T, M, or V.
The resulting constructs can be routinely screened for activities or functions
described
throughout the specification and known in the art. Preferably, the resulting
constructs have
an increased D-SLAM activity or function, while the remaining D-SLAM
activities or
functions are maintained. More preferably, the resulting constructs have more
than one
increased D-SLAM activity or function, while the remaining D-SLAM activities
or functions
are maintained.
Besides conservative amino acid substitution, variants of D-SLAM include (i)
substitutions with one or more of the non-conserved amino acid residues, where
the
substituted amino acid residues may or may not be one encoded by the genetic
code, or (ii)
substitution with one or more of amino acid residues having a substituent
group, or (iii)
fusion of the mature polypeptide with another compound, such as a compound to
increase the
stability and/or solubility of the polypeptide (for example, polyethylene
glycol), or (iv) fusion
of the polypeptide with additional amino acids, such as an IgG Fc fusion
region peptide, or
leader or secretory sequence, or a sequence facilitating purification. Such
variant
polypeptides are deemed to be within the scope of those skilled in the art
from the teachings
herein.
CA 02382659 2002-O1-29
WO 01/11046 PCT/US00/21130
34
For example, D-SLAM polypeptide variants containing amino acid substitutions
of
charged amino acids with other charged or neutral amino acids may produce
proteins with
improved characteristics, such as less aggregation. Aggregation of
pharmaceutical
formulations both reduces activity and increases clearance due to the
aggregate's
immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 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).)
For example, preferred non-conservative substitutions of D-SLAM include: M1
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V2 replaced with D, E, H,
K, R, N, Q, F,
W, Y, P, or C; M3 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R4
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; PS replaced with D, E, H,
K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; L6 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; W7
9
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S8
replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; L9 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L 10
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L11 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; W12 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; E13
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A14
replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L 15 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C;
L 16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P 17 replaced with
D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; I18 replaced with D, E, H, K, R, N,
Q, F, W, Y, P,
or C; T19 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V20 replaced
with D, E, H, K,
R, N, Q, F, W, Y, P, or C; T21 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G22
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A23 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; Q24 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C;
V25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L26 replaced with D,
E, H, K, R, N,
Q, F, W, Y, P, or C; S27 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K28 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V29 replaced with
D, E, H, K, R, N,
Q, F, W, Y, P, or C; G30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G31 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S32 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; V33 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L34 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; L35 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; V36
CA 02382659 2002-O1-29
WO 01/11046 PCT/US00/21130
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A37 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; A38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R39
replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P40 replaced with D, E,
H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; P41 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V,
5 N, Q, F, W, Y, or C; G42 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; F43 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q44 replaced with
D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; V45 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or
C; R46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E47
replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A48 replaced with D,
E, H, K, R, N,
10 Q, F, W, Y, P, or C; I49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; W50 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R51 replaced with
D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; S52 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; W54 replaced with D,
E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; P55 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V,
15 N, Q, F, W, Y, or C; S56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E57 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E58 replaced
with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L59 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; L60 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A61 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; F63
20 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F64
replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R65 replaced with D, E, A, G, I,
L, S, T, M, V, N,
Q, F, W, Y, P, or C; G66 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S67 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L68 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; E69 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; T70
25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L71 replaced with D,
E, H, K, R, N, Q,
F, W, Y, P, or C; Y72 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; H73
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S74
replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; R75 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y,
P, or C; F76 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or
C; L77 replaced
30 with D, E, H, K, R, N, Q, F, W, Y, P, or C; G78 replaced with D, E, H, K,
R, N, Q, F, W, Y,
P, or C; R79 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; A80 replaced
CA 02382659 2002-O1-29
WO 01/11046 PCT/US00/21130
36
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q81 replaced with D, E, H, K, R,
A, G, I, L, S, T,
M, V, F, W, Y, P, or C; L82 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; H83
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S84
replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; N85 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W,
Y, P, or C; L86 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S87
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L88 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; L90
replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; G91 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; L93 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E94 replaced with H, K, R, A, G,
I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; S95 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; D97 replaced with H, K,
R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; S98 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
G99 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N 100 replaced with
D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; F101 replaced with D, E, H, K, R, N,
Q, A, G, I, L,
S, T, M, V, P, or C; S 102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; V 103 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L 104 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; M 105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V 106
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; D107 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F,
W, Y, P, or C; T 108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R
109 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 6110 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; Q111 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C;
P112 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
W113 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T114 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; Q115 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P,
or C; T116 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L117 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; Q 118 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W,
Y, P, or C; L119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K120
replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V 121 replaced with D, E,
H, K, R, N, Q, F,
W, Y, P, or C; Y122 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; D123
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A124
replaced with D,
CA 02382659 2002-O1-29
WO 01/11046 PCT/US00/21130
37
E, H, K, R, N, Q, F, W, Y, P, or C; V 125 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C;
P 126 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
C; 8127 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P 128 replaced with
D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V 129 replaced with D, E, H, K,
R, N, Q, F, W, Y,
P, or C; V 130 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q131
replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V 132 replaced with D, E,
H, K, R, N, Q, F,
W, Y, P, or C; F133 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; I134
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A135 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; V 136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E
137 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8138 replaced
with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D 139 replaced with H, K, R, A, G,
I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; A140 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Q141
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P142
replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S 143 replaced with
D, E, H, K, R, N,
Q, F, W, Y, P, or C; K144 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C;
T145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C146 replaced with
D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q147 replaced with D, E, H, K, R,
A, G, I, L, S,
T, M, V, F, W, Y, P, or C; V148 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; F149
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; S 151 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C;
C152 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
W153 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A154 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; P155 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W,
Y, or C; N156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,
or C; I157
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 158 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; E159 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C;
I160 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T161 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; Y162 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or
C; S 163 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W 164 replaced
with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; 8165 replaced with D, E, A, G, I, L,
S, T, M, V, N,
Q, F, W, Y, P, or C; 8166 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C;
CA 02382659 2002-O1-29
WO 01/11046 PCT/US00/21130
38
E167 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
T168 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T169 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; M 170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D 171
replaced with H, K,
R, A, G, I, L, S, T, M, V, 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; G 173 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; M 174
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E175 replaced with H, K,
R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; P176 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V,
N, Q, F, W, Y, or C; H177 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C;
S 178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L 179 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; F180 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or
C; T181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D182 replaced
with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G 183 replaced with D, E, H, K,
R, N, Q, F, W, Y,
P, or C; Q 184 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; V 185
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L 186 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; S 187 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I188 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S 189 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; L190 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; P192 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N,
Q, F, W, Y, or C; G 193 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D
194 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, 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; D196 replaced with H, K, R, A, G, I,
L, S, T, M, V,
N, Q, F, W, Y, P, or C; V 197 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A 198
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y 199 replaced with D, E,
H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; 5200 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
C201 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
I202 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V203 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; 5204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N205
replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P206 replaced with D, E, H,
K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; V207 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or
C; 5208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W209 replaced
with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; D210 replaced with H, K, R, A, G, I,
L, S, T, M, V,
CA 02382659 2002-O1-29
WO 01/11046 PCT/US00/21130
39
N, Q, F, W, Y, P, or C; L211 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A212
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T213 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; V214 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T215 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P216 replaced with D, E, H, K, R,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; W217 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M,
V, P, or C; D218 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; S219
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C220 replaced with D, E,
H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; H221 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q,
F, W, Y, P, or C; H222 replaced with D, E, 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;
A224 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A225 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; P226 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, or C; 6227
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K228 replaced with D, E,
A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; A229 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
S230 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y231 replaced with
D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; K232 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q,
F, W, Y, P, or C; D233 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C;
V234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L235 replaced with
D, E, H, K, R,
N, Q, F, W, Y, P, or C; L236 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; V237
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V238 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; V239 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P240 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V241 replaced
with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S242 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L243
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L244 replaced with D, E,
H, K, R, N, Q,
F, W, Y, P, or C; L245 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
M246 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L247 replaced with D, E, H, K, R,
N, Q, F, W, Y,
P, or C; V248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T249
replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L250 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F251 replaced with D, E, H, K, R,
N, Q, A, G, I,
L, S, T, M, V, P, or C; S252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A253
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W254 replaced with D, E,
H, K, R, N, Q,
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WO 01/11046 PCT/US00/21130
A, G, I, L, S, T, M, V, P, or C; H255 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W,
Y, P, or C; W256 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; C257
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P258
replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C259 replaced with
D, E, H, K, R,
5 A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 5260 replaced with D, E, H, K,
R, N, Q, F, W, Y,
P, or C; 6261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K262
replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K263 replaced with D, E, A, G,
I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; K264 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P,
or C; K265 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
D266 replaced
10 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V267 replaced
with D, E, H, K,
R, N, Q, F, W, Y, P, or C; H268 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P,
or C; A269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D270 replaced
with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 8271 replaced with D, E, A, G,
I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; V272 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; 6273
15 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P274 replaced with D,
E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; E275 replaced with H, K, R, A, G, I, L,
S, T, M, V, N,
Q, F, W, Y, P, or C; T276 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E277 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N278 replaced
with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P279 replaced with D, E, H, K, R,
A, G, I, L, S,
20 T, M, V, N, Q, F, W, Y, or C; L280 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C;
V281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q282 replaced with
D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; D283 replaced with H, K, R, 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; P285
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C.
25 The resulting constructs can be routinely screened for activities or
functions described
throughout the specification and known in the art. Preferably, the resulting
constructs have
loss of a D-SLAM activity or function, while the remaining D-SLAM activities
or functions
are maintained. More preferably, the resulting constructs have more than one
loss of D
SLAM activity or function, while the remaining D-SLAM activities or functions
are
30 maintained.
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WO 01/11046 PCT/US00/21130
41
Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and 10)
can be
replaced with the substituted amino acids as described above (either
conservative or
nonconservative).
A further embodiment of the invention relates to a polypeptide which
comprises, or
alternatively consists of, the amino acid sequence of a D-SLAM polypeptide
having an amino
acid sequence which contains at least one amino acid substitution, but not
more than 50
amino acid substitutions, even more preferably, not more than 40 amino acid
substitutions,
still more preferably, not more than 30 amino acid substitutions, and still
even more
preferably, not more than 20 amino acid substitutions. Of course, in order of
ever-increasing
preference, it is highly preferable for a peptide or polypeptide to have an
amino acid sequence
which comprises, or alternatively consists of, the amino acid sequence of a D-
SLAM
polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5,
4, 3, 2 or 1 amino
acid substitutions. In specific embodiments, the number of additions,
substitutions, and/or
deletions in the amino acid sequence of Figures lA-1D or fragments thereof
(e.g., the mature
form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50
or 50-150,
conservative amino acid substitutions are preferable.
Polynucleotide and Polypeptide Fragments
The present invention is further directed to fragments of the isolated nucleic
acid
molecules described herein. By a fragment of an isolated nucleic acid molecule
having, for
example, the nucleotide sequence of the deposited cDNA (clone HDPJ039), a
nucleotide
sequence encoding the polypeptide sequence encoded by the deposited cDNA, a
nucleotide
sequence encoding the polypeptide sequence depicted in Figure 1 (SEQ ID N0:2),
the
nucleotide sequence shown in Figure 1 (SEQ ID NO: l ), or the complementary
strand thereto,
is intended fragments at least 15 nt, and more preferably at least about 20
nt, still more
preferably at least 30 nt, and even more preferably, at least about 40, 50,
100, 150, 200, 250,
300, 325, 350, 375, 400, 450, 500, 550, or 600 nt in length. These fragments
have numerous
uses that include, but are not limited to, diagnostic probes and primers as
discussed herein.
Of course, larger fragments, such as those of 501-1500 nt in length are also
useful according
to the present invention as are fragments corresponding to most, if not all,
of the nucleotide
sequences of the deposited cDNA (clone HDPJ039) or as shown in Figure 1 (SEQ
ID NO:1).
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42
By a fragment at least 20 nt in length, for example, is intended fragments
which include 20 or
more contiguous bases from, for example, the nucleotide sequence of the
deposited cDNA, or
the nucleotide sequence as shown in Figure 1 (SEQ ID NO:1 ).
Moreover, representative examples of D-SLAM polynucleotide fragments include,
for
example, fragments having a sequence from about nucleotide number 1-50, 51-
100, 101-150,
151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-
600, 651-
700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-
1100, 1101-
1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-
1500,
1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,
1851-
1900, 1901-1950, 1951-2000, and/or 2001 to the end of SEQ ID NO:1 or the
complementary
strand thereto, or the cDNA contained in the deposited clone. In this context
"about" includes
the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or
1) nucleotides, at
either terminus or at both termini.
Preferably, the polynucleotide fragments of the invention encode a polypeptide
which
demonstrates a D-SLAM functional activity. By a polypeptide demonstrating a D-
SLAM
"functional activity" is meant, a polypeptide capable of displaying one or
more known
functional activities associated with a full-length (complete) D-SLAM protein.
Such
functional activities include, but are not limited to, biological activity,
antigenicity [ability to
bind (or compete with a D-SLAM polypeptide for binding) to an anti-D-SLAM
antibody],
immunogenicity (ability to generate antibody which binds to a D-SLAM
polypeptide), ability
to form multimers with D-SLAM polypeptides of the invention, and ability to
bind to a
receptor or ligand for a D-SLAM polypeptide.
The functional activity of D-SLAM polypeptides, and fragments, variants
derivatives,
and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind
or
compete with full-length D-SLAM polypeptide for binding to anti-D-SLAM
antibody,
various immunoassays known in the art can be used, including but not limited
to, competitive
and non-competitive assay systems using techniques such as radioimmunoassays,
ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric
assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ
immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example), western
blots,
CA 02382659 2002-O1-29
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43
precipitation reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination
assays), complement fixation assays, immunofluorescence assays, protein A
assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody binding is
detected by
detecting a label on the primary antibody. In another embodiment, the primary
antibody is
detected by detecting binding of a secondary antibody or reagent to the
primary antibody. In
a further embodiment, the secondary antibody is labeled. Many means are known
in the art
for detecting binding in an immunoassay and are within the scope of the
present invention.
In another embodiment, where a D-SLAM ligand is identified, or the ability of
a
polypeptide fragment, variant or derivative of the invention to multimerize is
being evaluated,
binding can be assayed, e.g., by means well-known in the art, such as, for
example, reducing
and non-reducing gel chromatography, protein affinity chromatography, and
affinity blotting.
See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123. In
another embodiment,
physiological correlates of D-SLAM binding to its substrates (signal
transduction) can be
assayed.
In addition, assays described herein (see Examples) and otherwise known in the
art
may routinely be applied to measure the ability of D-SLAM polypeptides and
fragments,
variants derivatives and analogs thereof to elicit D-SLAM related biological
activity (either in
vitro or in vivo). Other methods will be known to the skilled artisan and are
within the scope
of the invention.
The present invention is further directed to fragments of the D-SLAM
polypeptide
described herein. By a fragment of an isolated the D-SLAM polypeptide, for
example,
encoded by the deposited cDNA (clone HDPJ039), the polypeptide sequence
encoded by the
deposited cDNA, the polypeptide sequence depicted in Figure 1 (SEQ ID N0:2),
is intended
to encompass polypeptide fragments contained in SEQ ID N0:2 or encoded by the
cDNA
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 as a
single continuous region. Representative examples of polypeptide fragments of
the
invention, include, for example, fragments from about amino acid number 1-20,
21-40, 41-
60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-
240, 241-
260, 261-280, or 281 to the end of the coding region. Moreover, polypeptide
fragments can
be at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150
amino acids in
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WO 01/11046 PCT/US00/21130
44
length. In this context "about" includes the particularly recited ranges,
larger or smaller by
several (5, 4, 3, 2, or 1 ) amino acids, at either extreme or at both
extremes.
Even if deletion of one or more amino acids from the N-terminus of a protein
results
in modification of loss of one or more biological functions of the protein,
other functional
activities (e.g., biological activities, ability to multimerize, ability to
bind D-SLAM ligand)
may still be retained. For example, the ability of shortened D-SLAM muteins to
induce
and/or bind to antibodies which recognize the complete or mature forms of the
polypeptides
generally will be retained when less than the majority of the residues of the
complete or
mature 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 an D-SLAM mutein with a large number of deleted N-
terminal
amino acid residues may retain some biological or immunogenic activities. In
fact, peptides
composed of as few as six D-SLAM amino acid residues may often evoke an immune
response.
Accordingly, polypeptide fragments include the secreted D-SLAM protein as well
as
the mature form. Further preferred polypeptide fragments include the secreted
D-SLAM
protein or the mature form having a continuous series of deleted residues from
the amino or
the carboxy terminus, or both. For example, any number of amino acids, ranging
from 1-60,
can be deleted from the amino terminus of either the secreted D-SLAM
polypeptide or the
mature form. Similarly, any number of amino acids, ranging from 1-30, can be
deleted from
the carboxy terminus of the secreted D-SLAM protein or mature form.
Furthermore, any
combination of the above amino and carboxy terminus deletions are preferred.
Similarly,
polynucleotide fragments encoding these D-SLAM polypeptide fragments are also
preferred.
The present invention is also directed to nucleic acid molecules comprising,
or alternatively,
consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%,
96%, 97%,
98% or 99% identical to the polynucleotide sequence encoding the D-SLAM
polypeptides
described above. The present invention also encompasses the above
polynucleotide
sequences fused to a heterologous polynucleotide sequence. Polypeptides
encoded by these
nucleic acids and/or polynucleotide sequences are also encompassed by the
invention, as are
polypeptides comprising, or alternatively consisting of, an amino acid
sequence at least 80%,
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WO 01/11046 PCT/US00/21130
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
described above, and polynucleotides that encode such polypeptides.
Particularly, N-terminal deletions of the D-SLAM polypeptide can be described
by
the general formula m-285, where m is an integer from 2 to 280, where m
corresponds to the
5 position of the amino acid residue identified in SEQ ID N0:2. More in
particular, the
invention provides polynucleotides encoding polypeptides comprising, or
alternatively
consisting of, an amino acid sequence selected from the group consisting of
residues V-2 to
P-285; M-3 to P-285; R-4 to P-285; P-5 to P-285; L-6 to P-285; W-7 to P-285; S-
8 to P-285;
L-9 to P-285; L-10 to P-285; L-11 to P-285; W-12 to P-285; E-13 to P-285; A-14
to P-285;
10 L-15 to P-285; L-16 to P-285; P-17 to P-285; I-18 to P-285; T-19 to P-285;
V-20 to P-285; T-
21 to P-285; G-22 to P-285; A-23 to P-285; Q-24 to P-285; V-25 to P-285; L-26
to P-285; S-
27 to P-285; K-28 to P-285; V-29 to P-285; G-30 to P-285; G-31 to P-285; S-32
to P-285; V-
33 to P-285; L-34 to P-285; L-35 to P-285; V-36 to P-285; A-37 to P-285; A-38
to P-285; R-
39 to P-285; P-40 to P-285; P-41 to P-285; G-42 to P-285; F-43 to P-285; Q-44
to P-285; V-
15 45 to P-285; R-46 to P-285; E-47 to P-285; A-48 to P-285; I-49 to P-285; W-
50 to P-285; R-
51 to P-285; S-52 to P-285; L-53 to P-285; W-54 to P-285; P-55 to P-285; S-56
to P-285; E-
57 to P-285; E-58 to P-285; L-59 to P-285; L-60 to P-285; A-61 to P-285; T-62
to P-285; F-
63 to P-285; F-64 to P-285; R-65 to P-285; G-66 to P-285; S-67 to P-285; L-68
to P-285; E-
69 to P-285; T-70 to P-285; L-71 to P-285; Y-72 to P-285; H-73 to P-285; S-74
to P-285; R-
20 75 to P-285; F-76 to P-285; L-77 to P-285; G-78 to P-285; R-79 to P-285; A-
80 to P-285; Q-
81 to P-285; L-82 to P-285; H-83 to P-285; S-84 to P-285; N-85 to P-285; L-86
to P-285; S-
87 to P-285; L-88 to P-285; E-89 to P-285; L-90 to P-285; G-91 to P-285; P-92
to P-285; L-
93 to P-285; E-94 to P-285; S-95 to P-285; G-96 to P-285; D-97 to P-285; S-98
to P-285; G-
99 to P-285; N-100 to P-285; F-101 to P-285; S-102 to P-285; V-103 to P-285; L-
104 to P-
25 285; M-105 to P-285; V-106 to P-285; D-107 to P-285; T-108 to P-285; R-109
to P-285; G-
110 to P-285; Q-111 to P-285; P-112 to P-285; W-113 to P-285; T-114 to P-285;
Q-115 to P-
285; T-116 to P-285; L-117 to P-285; Q-118 to P-285; L-119 to P-285; K-120 to
P-285; V-
121 to P-285; Y-122 to P-285; D-123 to P-285; A-124 to P-285; V-125 to P-285;
P-126 to P-
285; R-127 to P-285; P-128 to P-285; V-129 to P-285; V-130 to P-285; Q-131 to
P-285; V-
30 132 to P-285; F-133 to P-285; I-134 to P-285; A-135 to P-285; V-136 to P-
285; E-137 to P-
285; R-138 to P-285; D-139 to P-285; A-140 to P-285; Q-141 to P-285; P-142 to
P-285; S-
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143 to P-285; K-144 to P-285; T-145 to P-285; C-146 to P-285; Q-147 to P-285;
V-148 to P-
285; F-149 to P-285; L-150 to P-285; S-151 to P-285; C-152 to P-285; W-153 to
P-285; A-
154 to P-285; P-155 to P-285; N-156 to P-285; I-157 to P-285; S-158 to P-285;
E-159 to P-
285; I-160 to P-285; T-161 to P-285; Y-162 to P-285; S-163 to P-285; W-164 to
P-285; R-
165 to P-285; R-166 to P-285; E-167 to P-285; T-168 to P-285; T-169 to P-285;
M-170 to P-
285; D-171 to P-285; F-172 to P-285; G-173 to P-285; M-174 to P-285; E-175 to
P-285; P-
176 to P-285; H-177 to P-285; S-178 to P-285; L-179 to P-285; F-180 to P-285;
T-181 to P-
285; D-182 to P-285; G-183 to P-285; Q-184 to P-285; V-185 to P-285; L-186 to
P-285; S-
187 to P-285; I-188 to P-285; S-189 to P-285; L-190 to P-285; G-191 to P-285;
P-192 to P-
285; G-193 to P-285; D-194 to P-285; R-195 to P-285; D-196 to P-285; V-197 to
P-285; A-
198 to P-285; Y-199 to P-285; S-200 to P-285; C-201 to P-285; I-202 to P-285;
V-203 to P-
285; S-204 to P-285; N-205 to P-285; P-206 to P-285; V-207 to P-285; S-208 to
P-285; W-
209 to P-285; D-210 to P-285; L-211 to P-285; A-212 to P-285; T-213 to P-285;
V-214 to P-
285; T-215 to P-285; P-216 to P-285; W-217 to P-285; D-218 to P-285; S-219 to
P-285; C-
220 to P-285; H-221 to P-285; H-222 to P-285; E-223 to P-285; A-224 to P-285;
A-225 to P-
285; P-226 to P-285; G-227 to P-285; K-228 to P-285; A-229 to P-285; S-230 to
P-285; Y-
231 to P-285; K-232 to P-285; D-233 to P-285; V-234 to P-285; L-235 to P-285;
L-236 to P-
285; V-237 to P-285; V-238 to P-285; V-239 to P-285; P-240 to P-285; V-241 to
P-285; 5-
242 to P-285; L-243 to P-285; L-244 to P-285; L-245 to P-285; M-246 to P-285;
L-247 to P-
285; V-248 to P-285; T-249 to P-285; L-250 to P-285; F-251 to P-285; S-252 to
P-285; A-
253 to P-285; W-254 to P-285; H-255 to P-285; W-256 to P-285; C-257 to P-285;
P-258 to
P-285; C-259 to P-285; S-260 to P-285; G-261 to P-285; K-262 to P-285; K-263
to P-285; K-
264 to P-285; K-265 to P-285; D-266 to P-285; V-267 to P-285; H-268 to P-285;
A-269 to P-
285; D-270 to P-285; R-271 to P-285; V-272 to P-285; G-273 to P-285; P-274 to
P-285; E-
275 to P-285; T-276 to P-285; E-277 to P-285; N-278 to P-285; P-279 to P-285;
and L-280 to
P-285 of SEQ ID N0:2. Polypeptides encoded by these polynucleotides are also
encompassed by the invention. The present invention is also directed to
nucleic acid
molecules comprising, or alternatively, consisting of, a polynucleotide
sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide
sequence
encoding the D-SLAM polypeptides described above. The present invention also
encompasses the above polynucleotide sequences fused to a heterologous
polynucleotide
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sequence. Polypeptides encoded by these nucleic acids and/or polynucleotide
sequences are
also encompassed by the invention, as are polypeptides comprising, or
alternatively
consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%,
97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that
encode such polypeptides.
Also as mentioned above, even if deletion of one or more amino acids from the
C-terminus of a protein results in modification of loss of one or more
biological functions of
the protein, other functional activities (e.g., biological activities, ability
to multimerize,
ability to bind D-SLAM ligand) may still be retained. For example the ability
of the
shortened D-SLAM mutein to induce and/or bind to antibodies which recognize
the complete
or mature forms of the polypeptide generally will be retained when less than
the majority of
the residues of the complete or mature 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 an D-SLAM
mutein with a large
number of deleted C-terminal amino acid residues may retain some biological or
immunogenic activities. In fact, peptides composed of as few as six D-SLAM
amino acid
residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or
more
residues deleted from the carboxy terminus of the amino acid sequence of the D-
SLAM
polypeptide shown in Figure 1 (SEQ ID N0:2), as described by the general
formula 1-n,
where n is an integer from 7 to 284, where n corresponds to the position of
amino acid
residue identified in SEQ ID N0:2. More in particular, the invention provides
polynucleotides encoding 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 D-283;
M-1 to Q-282; M-1 to V-281; M-1 to L-280; M-1 to P-279; M-1 to N-278; M-1 to E-
277; M-
1 to T-276; M-1 to E-275; M-1 to P-274; M-1 to G-273; M-1 to V-272; M-1 to R-
271; M-1 to
D-270; M-1 to A-269; M-1 to H-268; M-1 to V-267; M-1 to D-266; M-1 to K-265; M-
1 to K-
264; M-1 to K-263; M-1 to K-262; M-1 to G-261; M-1 to S-260; M-1 to C-259; M-1
to P-
258; M-1 to C-257; M-1 to W-256; M-1 to H-255; M-1 to W-254; M-1 to A-253; M-1
to S-
252; M-1 to F-251; M-1 to L-250; M-1 to T-249; M-1 to V-248; M-1 to L-247; M-1
to M-
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246; M-1 to L-245; M-1 to L-244; M-1 to L-243; M-1 to S-242; M-1 to V-241; M-1
to P-
240; M-1 to V-239; M-1 to V-238; M-1 to V-237; M-1 to L-236; M-1 to L-235; M-1
to V-
234; M-1 to D-233; M-1 to K-232; M-1 to Y-231; M-1 to S-230; M-1 to A-229; M-1
to K-
228; M-1 to G-227; M-1 to P-226; M-1 to A-225; M-1 to A-224; M-1 to E-223; M-1
to H-
222; M-1 to H-221; M-1 fo C-220; M-1 to S-219; M-1 to D-218; M-1 to W-217; M-1
to P-
216; M-1 to T-215; M-1 to V-214; M-1 to T-213; M-1 to A-212; M-1 to L-211; M-1
to D-
210; M-1 to W-209; M-1 to S-208; M-1 to V-207; M-1 to P-206; M-1 to N-205; M-1
to 5-
204; M-1 to V-203; M-1 to I-202; M-1 to C-201; M-1 to S-200; M-1 to Y-199; M-1
to A-
198; M-1 to V-197; M-1 to D-196; M-1 to R-195; M-1 to D-194; M-1 to G-193; M-1
to P-
192; M-1 to G-191; M-1 to L-190; M-1 to S-189; M-1 to I-188; M-1 to S-187; M-1
to L-186;
M-1 to V-185; M-1 to Q-184; M-1 to G-183; M-1 to D-182; M-1 to T-181; M-1 to F-
180; M-
1 to L-179; M-1 to S-178; M-1 to H-177; M-1 to P-176; M-1 to E-175; M-1 to M-
174; M-1
to G-173; M-1 to F-172; M-1 to D-171; M-1 to M-170; M-1 to T-169; M-1 to T-
168; M-1 to
E-167; M-1 to R-166; M-1 to R-165; M-1 to W-164; M-1 to S-163; M-1 to Y-162; M-
1 to T-
161; M-1 to I-160; M-1 to E-159; M-1 to S-158; M-1 to I-157; M-1 to N-156; M-1
to P-155;
M-1 to A-154; M-1 to W-153; M-1 to C-152; M-1 to S-151; M-1 to L-150; M-1 to F-
149; M-
1 to V-148; M-1 to Q-147; M-1 to C-146; M-1 to T-145; M-1 to K-144; M-1 to S-
143; M-1
to P-142; M-1 to Q-141; M-1 to A-140; M-1 to D-139; M-1 to R-138; M-1 to E-
137; M-1 to
V-136; M-1 to A-135; M-1 to I-134; M-1 to F-133; M-1 to V-132; M-1 to Q-131; M-
1 to V-
130; M-1 to V-129; M-1 to P-128; M-1 to R-127; M-1 to P-126; M-1 to V-125; M-1
to A-
124; M-1 to D-123; M-1 to Y-122; M-1 to V-121; M-1 to K-120; M-1 to L-119; M-1
to Q-
118; M-1 to L-117; M-1 to T-116; M-1 to Q-115; M-1 to T-114; M-1 to W-113; M-1
to P-
112; M-1 to Q-111; M-1 to G-110; M-1 to R-109; M-1 to T-108; M-1 to D-107; M-1
to V-
106; M-1 to M-105; M-1 to L-104; M-1 to V-103; M-1 to S-102; M-1 to F-101; M-1
to N-
100; M-1 to G-99; M-1 to S-98; M-1 to D-97; M-1 to G-96; M-1 to S-95; M-1 to E-
94; M-1
to L-93; M-1 to P-92; M-1 to G-91; M-1 to L-90; M-1 to E-89; M-1 to L-88; M-1
to S-87; M-
1 to L-86; M-1 to N-85; M-1 to S-84; M-1 to H-83; M-1 to L-82; M-1 to Q-81; M-
1 to A-80;
M-1 to R-79; M-1 to G-78; M-1 to L-77; M-1 to F-76; M-1 to R-75; M-1 to S-74;
M-1 to H-
73; M-1 to Y-72; M-1 to L-71; M-1 to T-70; M-1 to E-69; M-1 to L-68; M-1 to S-
67; M-1 to
G-66; M-1 to R-65; M-1 to F-64; M-1 to F-63; M-1 to T-62; M-1 to A-61; M-1 to
L-60; M-1
to L-59; M-1 to E-58; M-1 to E-57; M-1 to S-56; M-1 to P-55; M-1 to W-54; M-1
to L-53;
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M-1 to S-52; M-1 to R-51; M-1 to W-50; M-1 to I-49; M-1 to A-48; M-1 to E-47;
M-1 to R-
46; M-1 to V-45; M-1 to Q-44; M-1 to F-43; M-1 to G-42; M-1 to P-41; M-1 to P-
40; M-1 to
R-39; M-1 to A-38; M-1 to A-37; M-1 to V-36; M-1 to L-35; M-1 to L-34; M-1 to
V-33; M-1
to S-32; M-1 to G-31; M-1 to G-30; M-1 to V-29; M-1 to K-28; M-1 to S-27; M-1
to L-26;
M-1 to V-25; M-1 to Q-24; M-1 to A-23; M-1 to G-22; M-1 to T-21; M-1 to V-20;
M-1 to T-
19; M-1 to I-18; M-1 to P-17; M-1 to L-16; M-1 to L-15; M-1 to A-14; M-1 to E-
13; M-1 to
W-12; M-1 to L-11; M-1 to L-10; M-1 to L-9; M-1 to S-8; and M-1 to W-7 of SEQ
ID N0:2.
Polypeptides encoded by these polynucleotides are also encompassed by the
invention. The
present invention is also directed to nucleic acid molecules comprising, or
alternatively,
consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%,
96%, 97%,
98% or 99% identical to the polynucleotide sequence encoding the D-SLAM
polypeptides
described above. The present invention also encompasses the above
polynucleotide
sequences fused to a heterologous polynucleotide sequence. Polypeptides
encoded by these
nucleic acids and/or polynucleotide sequences are also encompassed by the
invention, as are
polypeptides comprising, or alternatively consisting of, an amino acid
sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
described above, and polynucleotides that encode such polypeptides.
In addition, any of the above listed N- or C-terminal deletions can be
combined to
produce a N- and C-terminal deleted D-SLAM polypeptide. The invention also
provides
polypeptides having one or more amino acids deleted from both the amino and
the carboxyl
termini, which may be described generally as having residues m-n of SEQ ID
N0:2, where n
and m are integers as described above. Polynucleotides encoding these
polypeptides are also
encompassed by the invention.
Moreover, preferred N- and C-terminal deletion mutants comprise, or in the
alternative consist of, the predicted secreted form of D-SLAM. Preferred
secreted forms of
the D-SLAM include polypeptides comprising, or alternatively consisting of, an
amino acid
sequence selected from the group consisting of residues M-1 to K-232; V-2 to K-
232; M-3 to
K-232; R-4 to K-232; P-5 to K-232; L-6 to K-232; W-7 to K-232; S-8 to K-232; L-
9 to K-
232; L-10 to K-232; L-11 to K-232; W-12 to K-232; E-13 to K-232; A-14 to K-
232; L-15 to
K-232; L-16 to K-232; P-17 to K-232; I-18 to K-232; T-19 to K-232; V-20 to K-
232; T-21 to
K-232; G-22 to K-232; A-23 to K-232; Q-24 to K-232; V-25 to K-232; L-26 to K-
232; S-27
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to K-232; K-28 to K-232; V-29 to K-232; G-30 to K-232; G-31 to K-232; S-32 to
K-232; V-
33 to K-232; L-34 to K-232; L-35 to K-232; V-36 to K-232; A-37 to K-232; A-38
to K-232;
R-39 to K-232; P-40 to K-232; P-41 to K-232; G-42 to K-232; F-43 to K-232; Q-
44 to K-
232; V-45 to K-232; R-46 to K-232; E-47 to K-232; A-48 to K-232; I-49 to K-
232; W-50 to
5 K-232; R-51 to K-232; S-52 to K-232; L-53 to K-232; W-54 to K-232; P-55 to K-
232; S-56
to K-232; E-57 to K-232; E-58 to K-232; L-59 to K-232; L-60 to K-232; A-61 to
K-232; T-
62 to K-232; F-63 to K-232; F-64 to K-232; R-65 to K-232; G-66 to K-232; S-67
to K-232;
L-68 to K-232; E-69 to K-232; T-70 to K-232; L-71 to K-232; Y-72 to K-232; H-
73 to K-
232; S-74 to K-232; R-75 to K-232; F-76 to K-232; L-77 to K-232; G-78 to K-
232; R-79 to
10 K-232; A-80 to K-232; Q-81 to K-232; L-82 to K-232; H-83 to K-232; S-84 to
K-232; N-85
to K-232; L-86 to K-232; S-87 to K-232; L-88 to K-232; E-89 to K-232; L-90 to
K-232; G-
91 to K-232; P-92 to K-232; L-93 to K-232; E-94 to K-232; S-95 to K-232; G-96
to K-232;
D-97 to K-232; S-98 to K-232; G-99 to K-232; N-100 to K-232; F-101 to K-232; S-
102 to K-
232; V-103 to K-232; L-104 to K-232; M-105 to K-232; V-106 to K-232; D-107 to
K-232; T-
15 108 to K-232; R-109 to K-232; G-110 to K-232; Q-111 to K-232; P-112 to K-
232; W-113 to
K-232; T-114 to K-232; Q-115 to K-232; T-116 to K-232; L-117 to K-232; Q-118
to K-232;
L-119 to K-232; K-120 to K-232; V-121 to K-232; Y-122 to K-232; D-123 to K-
232;. A-124
to K-232; V-125 to K-232; P-126 to K-232; R-127 to K-232; P-128 to K-232; V-
129 to K-
232; V-130 to K-232; Q-131 to K-232; V-132 to K-232; F-133 to K-232; I-134 to
K-232; A-
20 135 to K-232; V-136 to K-232; E-137 to K-232; R-138 to K-232; D-139 to K-
232; A-140 to
K-232; Q-141 to K-232; P-142 to K-232; S-143 to K-232; K-144 to K-232; T-145
to K-232;
C-146 to K-232; Q-147 to K-232; V-148 to K-232; F-149 to K-232; L-150 to K-
232; S-151
to K-232; C-152 to K-232; W-153 to K-232; A-154 to K-232; P-155 to K-232; N-
156 to K-
232; I-157 to K-232; S-158 to K-232; E-159 to K-232; I-160 to K-232; T-161 to
K-232; Y-
25 162 to K-232; S-163 to K-232; W-164 to K-232; R-165 to K-232; R-166 to K-
232; E-167 to
K-232; T-168 to K-232; T-169 to K-232; M-170 to K-232; D-171 to K-232; F-172
to K-232;
G-173 to K-232; M-174 to K-232; E-175 to K-232; P-176 to K-232; H-177 to K-
232; S-178
to K-232; L-179 to K-232; F-180 to K-232; T-181 to K-232; D-182 to K-232; G-
183 to K-
232; Q-184 to K-232; V-185 to K-232; L-186 to K-232; S-187 to K-232; I-188 to
K-232; S-
30 189 to K-232; L-190 to K-232; G-191 to K-232; P-192 to K-232; G-193 to K-
232; D-194 to
K-232; R-195 to K-232; D-196 to K-232; V-197 to K-232; A-198 to K-232; Y-199
to K-232;
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S-200 to K-232; C-201 to K-232; I-202 to K-232; V-203 to K-232; S-204 to K-
232; N-205 to
K-232; P-206 to K-232; V-207 to K-232; S-208 to K-232; W-209 to K-232; D-210
to K-232;
L-211 to K-232; A-212 to K-232; T-213 to K-232; V-214 to K-232; T-215 to K-
232; P-216
to K-232; W-217 to K-232; D-218 to K-232; S-219 to K-232; C-220 to K-232; H-
221 to K-
232; H-222 to K-232; E-223 to K-232; A-224 to K-232; A-225 to K-232; P-226 to
K-232;
and G-227 to K-232 of SEQ ID N0:2. Polypeptides encoded by these
polynucleotides are
also encompassed by the invention. The present invention is also directed to
nucleic acid
molecules comprising, or alternatively, consisting of, a polynucleotide
sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide
sequence
encoding the D-SLAM polypeptides described above. The present invention also
encompasses the above polynucleotide sequences fused to a heterologous
polynucleotide
sequence. Polypeptides encoded by these nucleic acids and/or polynucleotide
sequences are
also encompassed by the invention, as are polypeptides comprising, or
alternatively
consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%,
97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that
encode such polypeptides.
Additionally, preferred N- and C-terminal deletion mutants comprise, or in the
alternative consist of, fragments lacking the predicted signal sequence of D-
SLAM. Preferred
fragments of D-SLAM include polypeptides comprising, or alternatively
consisting of, an
amino acid sequence selected from the group consisting of residues A-23 to L-
284; A-23 to
D-283; A-23 to Q-282; A-23 to V-281; A-23 to L-280; A-23 to P-279; A-23 to N-
278; A-23
to E-277; A-23 to T-276; A-23 to E-275; A-23 to P-274; A-23 to G-273; A-23 to
V-272; A-
23 to R-271; A-23 to D-270; A-23 to A-269; A-23 to H-268; A-23 to V-267; A-23
to D-266;
A-23 to K-265; A-23 to K-264; A-23 to K-263; A-23 to K-262; A-23 to G-261; A-
23 to S-
260; A-23 to C-259; A-23 to P-258; A-23 to C-257; A-23 to W-256; A-23 to H-
255; A-23 to
W-254; A-23 to A-253; A-23 to S-252; A-23 to F-251; A-23 to L-250; A-23 to T-
249; A-23
to V-248; A-23 to L-247; A-23 to M-246; A-23 to L-245; A-23 to L-244; A-23 to
L-243; A-
23 to S-242; A-23 to V-241; A-23 to P-240; A-23 to V-239; A-23 to V-238; A-23
to V-237;
A-23 to L-236; A-23 to L-235; A-23 to V-234; A-23 'to D-233; A-23 to K-232; A-
23 to Y-
231; A-23 to S-230; A-23 to A-229; A-23 to K-228; A-23 to G-227; A-23 to P-
226; A-23 to
A-225; A-23 to A-224; A-23 to E-223; A-23 to H-222; A-23 to H-221; A-23 to C-
220; A-23
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to S-219; A-23 to D-218; A-23 to W-217; A-23 to P-216; A-23 to T-215; A-23 to
V-214; A-
23 to T-213; A-23 to A-212; A-23 to L-211; A-23 to D-210; A-23 to W-209; A-23
to S-208;
A-23 to V-207; A-23 to P-206; A-23 to N-205; A-23 to S-204; A-23 to V-203; A-
23 to I-202;
A-23 to C-201; A-23 to S-200; A-23 to Y-199; A-23 to A-198; A-23 to V-197; A-
23 to D-
196; A-23 to R-195; A-23 to D-194; A-23 to G-193; A-23 to P-192; A-23 to G-
191; A-23 to
L-190; A-23 to S-189; A-23 to I-188; A-23 to S-187; A-23 to L-186; A-23 to V-
185; A-23 to
Q-184; A-23 to G-183; A-23 to D-182; A-23 to T-181; A-23 to F-180; A-23 to L-
179; A-23
to S-178; A-23 to H-177; A-23 to P-176; A-23 to E-175; A-23 to M-174; A-23 to
G-173; A-
23 to F-172; A-23 to D-171; A-23 to M-170; A-23 to T-169; A-23 to T-168; A-23
to E-167;
A-23 to R-166; A-23 to R-165; A-23 to W-164; A-23 to S-163; A-23 to Y-162; A-
23 to T-
161; A-23 to I-160; A-23 to E-159; A-23 to S-158; A-23 to I-157; A-23 to N-
156; A-23 to P-
155; A-23 to A-154; A-23 to W-153; A-23 to C-152; A-23 to S-151; A-23 to L-
150; A-23 to
F-149; A-23 to V-148; A-23 to Q-147; A-23 to C-146; A-23 to T-145; A-23 to K-
144; A-23
to S-143; A-23 to P-142; A-23 to Q-141; A-23 to A-140; A-23 to D-139; A-23 to
R-138; A-
23 to E-137; A-23 to V-136; A-23 to A-135; A-23 to I-134; A-23 to F-133; A-23
to V-132;
A-23 to Q-131; A-23 to V-130; A-23 to V-129; A-23 to P-128; A-23 to R-127; A-
23 to P-
126; A-23 to V-125; A-23 to A-124; A-23 to D-123; A-23 to Y-122; A-23 to V-
121; A-23 to
K-120; A-23 to L-119; A-23 to Q-118; A-23 to L-117; A-23 to T-116; A-23 to Q-
115; A-23
to T-114; A-23 to W-113; A-23 to P-112; A-23 to Q-111; A-23 to G-110; A-23 to
R-109; A-
23 to T-108; A-23 to D-107; A-23 to V-106; A-23 to M-105; A-23 to L-104; A-23
to V-103;
A-23 to S-102; A-23 to F-101; A-23 to N-100; A-23 to G-99; A-23 to S-98; A-23
to D-97; A-
23 to G-96; A-23 to S-95; A-23 to E-94; A-23 to L-93; A-23 to P-92; A-23 to G-
91; A-23 to
L-90; A-23 to E-89; A-23 to L-88; A-23 to S-87; A-23 to L-86; A-23 to N-85; A-
23 to S-84;
A-23 to H-83; A-23 to L-82; A-23 to Q-81; A-23 to A-80; A-23 to R-79; A-23 to
G-78; A-23
to L-77; A-23 to F-76; A-23 to R-75; A-23 to S-74; A-23 to H-73; A-23 to Y-72;
A-23 to L-
71; A-23 to T-70; A-23 to E-69; A-23 to L-68; A-23 to S-67; A-23 to G-66; A-23
to R-65; A-
23 to F-64; A-23 to F-63; A-23 to T-62; A-23 to A-61; A-23 to L-60; A-23 to L-
59; A-23 to
E-58; A-23 to E-57; A-23 to S-56; A-23 to P-55; A-23 to W-54; A-23 to L-53; A-
23 to S-52;
A-23 to R-51; A-23 to W-50; A-23 to I-49; A-23 to A-48; A-23 to E-47; A-23 to
R-46; A-23
to V-45; A-23 to Q-44; A-23 to F-43; A-23 to G-42; A-23 to P-41; A-23 to P-40;
A-23 to R-
39; A-23 to A-38; A-23 to A-37; A-23 to V-36; A-23 to L-35; A-23 to L-34; A-23
to V-33;
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A-23 to S-32; A-23 to G-31; A-23 to G-30; and A-23 to V-29 of SEQ ID N0:2.
Polypeptides
encoded by these polynucleotides are also encompassed by the invention. The
present
invention is also directed to nucleic acid molecules comprising, or
alternatively, consisting
of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
or 99%
identical to the polynucleotide sequence encoding the D-SLAM polypeptides
described
above. The present invention also encompasses the above polynucleotide
sequences fused to
a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic
acids and/or
polynucleotide sequences are also encompassed by the invention, as are
polypeptides
comprising, or alternatively consisting of, an amino acid sequence at least
80%, 85%, 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described
above,
and polynucleotides that encode such polypeptides.
The present application is also directed to proteins containing polypeptides
at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the D-SLAM
polypeptide
sequence set forth herein m-n. In preferred embodiments, the application is
directed to
proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or
99% identical to polypeptides having the amino acid sequence of the specific D-
SLAM N-
and C-terminal deletions recited herein. Polynucleotides encoded by these
polypeptides are
also encompassed by the invention. The present invention is also directed to
nucleic acid
molecules comprising, or alternatively, consisting of, a polynucleotide
sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide
sequence
encoding the D-SLAM polypeptides described above. The present invention also
encompasses the above polynucleotide sequences fused to a heterologous
polynucleotide
sequence. Polypeptides encoded by these nucleic acids and/or polynucleotide
sequences are
also encompassed by the invention, as are polypeptides comprising, or
alternatively
consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%,
97%, 98%
or 99% identical to the amino acid sequence described above, and
polynucleotides that
encode such polypeptides.
Among the especially preferred fragments of the invention are fragments
characterized by structural or functional attributes of D-SLAM. Such fragments
include
amino acid residues that comprise, or alternatively consist of, alpha-helix
and alpha-helix
forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions
("beta-
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54
regions"), turn and turn-forming regions ("turn-regions"), coil and coil-
forming regions
("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic
regions, beta
amphipathic regions, surface forming regions, and high antigenic index regions
(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
complete (i.e., full-length) D-SLAM (SEQ ID N0:2). Certain preferred regions
are those set
out in Figure 3 and include, but are not limited to, regions of the
aforementioned types
identified by analysis of the amino acid sequence depicted in Figure 1 (SEQ ID
N0:2), such
preferred regions include; Gamier-Robson predicted alpha-regions, beta-
regions, turn-
regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions,
turn-regions,
and coil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobic
regions; Eisenberg
alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson-
Wolf high
antigenic index regions, as predicted using the default parameters of these
computer
programs. Polynucleotides encoding these polypeptides are also encompassed by
the
invention.
In additional embodiments, the polynucleotides of the invention encode
functional
attributes of D-SLAM. Preferred embodiments of the invention in this regard
include
fragments that comprise, or alternatively consist of, alpha-helix and alpha-
helix forming
regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-
regions"), turn
and turn-forming regions ("turn-regions"), coil and coil-forming regions
("coil-regions"),
hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic
regions, flexible regions, surface-forming regions and high antigenic index
regions of D-
SLAM.
The data representing the structural or functional attributes of D-SLAM set
forth in
Figure 1 and/or Table I, as described above, was generated using the various
modules and
algorithms of the DNA*STAR set on default parameters. In a preferred
embodiment, the
data presented in columns VIII, IX, XIII, and XIV of Table I can be used to
determine
regions of D-SLAM 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 IV
by choosing values which represent regions of the polypeptide which are likely
to be exposed
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on the surface of the polypeptide in an environment in which antigen
recognition may occur
in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in Figure 3, but may,
as shown in
Table I, be represented or identified by using tabular representations of the
data presented in
5 Figure 3. The DNA*STAR computer algorithm used to generate Figure 3 (set on
the original
default parameters) was used to present the data in Figure 3 in a tabular
format (See Table I).
The tabular format of the data in Figure 3 may be used to easily determine
specific
boundaries of a preferred region.
The above-mentioned preferred regions set out in Figure 3 and in Table I
include, but
10 are not limited to, regions of the aforementioned types identified by
analysis of the amino
acid sequence set out in Figure 1. As set out in Figure 3 and in Table I, such
preferred
regions include 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,
15 Karplus-Schulz flexible regions, Emini surface-forming regions and Jameson-
Wolf regions of
high antigenic index.
Among highly preferred fragments in this regard are those that comprise, or
alternatively consist of, regions of D-SLAM that combine several structural
features, such as
several of the features set out in Table 1.
20 Other preferred fragments are biologically active D-SLAM fragments.
Biologically
active fragments are those exhibiting activity similar, but not necessarily
identical, to an
activity of the D-SLAM polypeptide. The biological activity of the fragments
may include an
improved desired activity, or a decreased undesirable activity.
However, many polynucleotide sequences, such as EST sequences, are publicly
25 available and accessible through sequence databases. Some of these
sequences are related to
SEQ ID NO:1 and may have been publicly available prior to conception of the
present
invention. Preferably, such related polynucleotides are specifically excluded
from the scope
of the present invention. For example, the following ESTs are preferably
excluded from the
present invention: AA917335; AI094818, AI298413; N62522; AA627522; 811635;
30 AA320408; AA379112; 809841; 220320; N79421; D45800; T98959; AA217290;
N30197;
AA286132; and AA633983 (hereby incorporated by reference in their entirety.)
However, to
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56
list every related sequence would be cumbersome. Accordingly, preferably
excluded from
the present invention are one or more polynucleotides comprising, or
alternatively consisting
of, a nucleotide sequence described by the general formula of a-b, where a is
any integer
between 1 to 3206 of SEQ ID NO:1, b is an integer of 15 to 3220, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and
where the b is
greater than or equal to a + 14.
Enitope-Bearing Portions
The present invention encompasses polypeptides comprising, or alternatively
consisting of, an epitope of the polypeptide having an amino acid sequence of
SEQ ID N0:2,
or an epitope of the polypeptide sequence encoded by a polynucleotide sequence
contained in
deposited clone HDPJ039 (ATCC Deposit No. 209623) or encoded by a
polynucleotide that
hybridizes to the complement of the sequence of SEQ ID NO:1 or contained in
deposited
clone HDPJ039 under stringent hybridization conditions or lower stringency
hybridization
conditions as defined supra. The present invention further encompasses
polynucleotide
sequences encoding an epitope of a polypeptide sequence of the invention (such
as, for
example, the sequence disclosed in SEQ ID NO:1 ), polynucleotide sequences of
the
complementary strand of a polynucleotide sequence encoding an epitope of the
invention,
and polynucleotide sequences which hybridize to the complementary strand under
stringent
hybridization conditions or lower stringency hybridization conditions defined
supra.
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 a
polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. 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
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57
does not necessarily exclude cross-reactivity with other antigens. Antigenic
epitopes need
not necessarily be immunogenic.
Fragments that 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).
In the present invention, antigenic epitopes 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 15, at
least 20, at least 25, and, most preferably, between about 15 to about 30
amino acids.
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.
Antigenic epitopes are useful, for example, to raise antibodies, including
monoclonal
antibodies, that specifically bind the epitope. 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)).
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). In one embodiment, a preferred immunogenic
epitope includes
the secreted protein. The polypeptides comprising one or more immunogenic
epitopes may be
presented for eliciting an antibody response together with a carrier protein,
such as an
albumin, to an animal system (such as, for example, 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 antibodies capable of binding to, at the
very least, linear
epitopes in a denatured polypeptide (e.g., in Western blotting).
Epitope-bearing polypeptides of the present invention 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
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keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides
containing
cysteine residues may be coupled to a carrier 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, for
example, 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 that 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.
As one of skill in the art will appreciate, and as discussed above, the
polypeptides of
the present invention comprising an immunogenic or antigenic epitope can be
fused to other
polypeptide sequences. For example, the polypeptides of the present invention
may be fused
with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions
thereof
(CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in
chimeric .
polypeptides. Such fusion proteins may facilitate purification and may
increase half life in
vivo. This has been shown for chimeric proteins 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 mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al.,
Nature,
331:84-86 ( 1988). IgG Fusion proteins that have a disulfide-linked dimeric
structure due to
the IgG portion desulfide 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
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59
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.
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 polypeptides of the invention, such methods can be used to generate
polypeptides with
altered activity, as well as'agonists and antagonists of the polypeptides.
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, alteration of
polynucleotides
corresponding to SEQ ID NO:1 and the polypeptides encoded by these
polynucleotides may
be achieved by DNA shuffling. DNA shuffling involves the assembly of two or
more DNA
segments by homologous or site-specific recombination to generate variation in
the
polynucleotide sequence. In another embodiment, polynucleotides of the
invention, or the
encoded polypeptides, 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 components, motifs, sections, parts, domains,
fragments, etc., of a
polynucleotide coding a polypeptide of the invention may be recombined with
one or more
components, motifs, sections, parts, domains, fragments, etc. of one or more
heterologous
molecules.
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen
receptors
(TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or
variant of
SEQ ID N0:2, and/or an epitope, of the present invention (as determined by
immunoassays
well known in the art for assaying specific antibody-antigen binding).
Antibodies of the
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invention include, but are not limited to, polyclonal, monoclonal,
multispecific, human,
humanized or chimeric antibodies, single chain antibodies, Fab fragments,
F(ab') fragments,
fragments produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies
(including, e.g., anti-id antibodies to antibodies of the invention), and
epitope-binding
5 fragments of any of the above. 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. The immunoglobulin molecules of the invention can be of any type
(e.g., IgG,
IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG3, IgG4, IgA 1 and
IgA2) or
10 subclass of immunoglobulin molecule. Immunoglobulins may have both a heavy
and light
chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired
with a light
chain of the kappa or lambda forms. In a specific embodiment, the
immunoglobulin
molecules of the invention are IgGI. In another specific embodiment, the
immunoglobulin
molecules of the invention are IgG4.
15 Most preferably the antibodies are human antigen-binding antibody fragments
of the
present invention and include, but are not limited to, Fab, Fab' and F(ab')2,
Fd, single-chain
Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising
either a VL or VH domain. Antigen-binding antibody fragments, including single-
chain
antibodies, may comprise the variable regions) alone or in combination with
the entirety or a
20 portion of the following: hinge region, CH1, CH2, and CH3 domains. Also
included in the
invention are antigen-binding fragments also comprising any combination of
variable
regions) with a hinge region, CH1, CH2, and CH3 domains. 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, ship rabbit, goat, guinea pig,
camel, horse, or
25 chicken. As used herein, "human" antibodies include antibodies having the
amino acid
sequence of a human immunoglobulin and include antibodies isolated from human
immunoglobulin libraries or from animals transgenic for one or more human
immunoglobulin
and that do not express endogenous immunoglobulins, as described infra and,
for example in,
U.S. Patent No. 5,939,598 by Kucherlapati et al.
30 The antibodies of the present invention may be monospecific, bispecific,
trispecific or
of greater multispecificity. Multispecific antibodies may be specific for
different epitopes of
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61
a polypeptide of the present invention or may be specific for both a
polypeptide of the present
invention as well as for a heterologous epitope, such as a heterologous
polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO 92/08802;
W091/00360;
WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos.
4,474,893;
S 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.
148:1547-1553
( 1992).
Antibodies of the present invention may be described or specified in terms of
the
epitope(s) or portions) of a polypeptide of the present invention which they
recognize or
specifically bind. The epitope(s) or polypeptide portions) may be specified as
described
herein, e.g., by N-terminal and C-terminal positions, by size in contiguous
amino acid
residues, or listed in the Tables and Figures. Antibodies which specifically
bind any epitope
or polypeptide of the present invention may also be excluded. Therefore, the
present
invention includes antibodies that specifically bind polypeptides of the
present invention, and
allows for the exclusion of the same.
In preferred, nonexclusive embodiments, the antibodies of the invention
inhibit one or
more biological activities of D-SLAM polypeptides of the invention through
specific binding.
In more preferred embodiments, the antibody of the invention inhibits D-SLAM-
mediated
inhibition of B cell proliferation. In additional preferred embodiments, the
antibody of the
invention stimulates B cell proliferation. In additional preferred
embodiments, the antibody
of the invention is used in combination with one or more additional antibodies
and/or
polypeptides and/or other agents to regulate B cell proliferation and/or
biological phenomena
related thereto.
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 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
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62
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). Antibodies of the present
invention may also
be described or specified in terms of their binding affinity to a polypeptide
of the invention.
Preferred binding affinities include those with a dissociation constant or Kd
less than 5 X 10-'
M, 10-z M, 5 X 10-' M, 10-' M, 5 X 10-'' M, 10-° M, 5 X 105 M, 10-5 M,
5 X 10-fi M, 10-fiM, 5 X
10-' M, 10' M, 5 X 10-8 M, 10-8 M, 5 X 10-y M, 10-y M, 5 X 10-'° M, 10-
"' M, 5 X 10-" M, 10-"
M, 5 X 10-'z M, 10'2 M, 5 X 10-'j M, 10-'3 M, 5 X 10-'4 M, 10-" M, 5 X 10-'S
M, or 10-'S M.
The invention also provides antibodies that competitively inhibit binding of
an
antibody to an epitope of the invention as determined by any method known in
the art for
determining competitive binding, for example, the immunoassays described
herein. In
preferred embodiments, the antibody competitively inhibits binding to the
epitope by at least
95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at
least 60%, or at
least 50%.
Antibodies of the present invention may act as agonists or antagonists of the
polypeptides of the present invention. For example, the present invention
includes antibodies
which disrupt the receptor/ligand interactions with the polypeptides of the
invention either
partially or fully. Preferrably, antibodies of the present invention bind an
antigenic epitope
disclosed herein, or a portion thereof. The invention features both receptor-
specific
antibodies and ligand-specific antibodies. The invention also features
receptor-specific
antibodies which do not prevent ligand binding but prevent receptor
activation. Receptor
activation (i.e., signaling) may be determined by techniques described herein
or otherwise
known in the art. For example, receptor activation can be determined by
detecting the
phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its
substrate by
immunoprecipitation followed by western blot analysis (for example, as
described supra). In
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specific embodiments, antibodies are provided that inhibit ligand activity or
receptor activity
by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at
least 70%, at least
60%, or at least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent
ligand
binding and receptor activation as well as antibodies that recognize the
receptor-ligand
complex, and, preferably, do not specifically recognize the unbound receptor
or the unbound
ligand. Likewise, included in the invention are neutralizing antibodies which
bind the ligand
and prevent binding of the ligand to the receptor, as well as antibodies which
bind the ligand,
thereby preventing receptor activation, but do not prevent the ligand from
binding the
receptor. Further included in the invention are antibodies which activate the
receptor. These
antibodies may act as receptor agonists, i.e., potentiate or activate either
all or a subset of the
biological activities of the ligand-mediated receptor activation, for example,
by inducing
dimerization of the receptor. The antibodies may be specified as agonists,
antagonists or
inverse agonists for biological activities comprising the specific biological
activities of the
peptides of the invention disclosed herein. The above antibody agonists can be
made using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998);
Zhu et al.,
Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179
(1998);
Prat et al., J. Cell. Sci. 111 (Pt2):237-247 ( 1998); Pitard et al., J.
Immunol. Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson
et al., J. Biol.
Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);
Muller
et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20
(1996) (which
are all incorporated by reference herein in their entireties).
Antibodies of the present invention may be used, for example, but not limited
to, to
purify, detect, and target the polypeptides of the present invention,
including both in vitro and
in vivn diagnostic and therapeutic methods. For example, the antibodies have
use in
immunoassays for qualitatively and quantitatively measuring levels of the
polypeptides of the
present invention 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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As discussed in more detail below, the antibodies of the present invention may
be
used either alone or in combination with other compositions. The antibodies
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.
The antibodies of the invention include derivatives that are modified, i.e, by
the
covalent attachment of any type of molecule to the antibody such that covalent
attachment
does not prevent the antibody from generating an anti-idiotypic response. For
example, but
not by way of limitation, the antibody derivatives include antibodies that
have been modified,
e.g., by glycosylation, acetylation, pegylation, phosphylation, 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.
The antibodies of the present invention may be generated by any suitable
method
known in the art. Polyclonal antibodies to an antigen-of-interest can be
produced by various
procedures well known in the art. For example, a polypeptide of the invention
can be
administered to various host animals including, but not limited to, rabbits,
mice, rats, etc. to
induce the production of sera containing polyclonal antibodies specific for
the antigen.
Various adjuvants may be used to increase the immunological response,
depending on the
host species, and include but are not limited to, Freund's (complete and
incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques known
in
the art including the use of hybridoma, recombinant, and phage display
technologies, or a
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combination thereof. For example, monoclonal antibodies can be produced using
hybridoma
techniques including those known in the art and taught, for example, in Harlow
et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988);
Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier,
5 N.Y., 1981) (said references incorporated by reference in their entireties).
The term
"monoclonal antibody" as used herein is not limited to antibodies produced
through
hybridoma technology. The term "monoclonal antibody" refers to an antibody
that is derived
from a single clone, including any eukaryotic, prokaryotic, or phage clone,
and not the
method by which it is produced.
10 A "monoclonal antibody" may comprise, or alternatively consist of, two
proteins, i.e.,
a heavy and a light chain.
Methods for producing and screening for specific antibodies using hybridoma
technology are routine and well known in the art and are discussed in detail
in the Examples
(e.g., Example 11). In a non-limiting example, mice can be immunized with a
polypeptide of
15 the invention or a cell expressing such peptide. Once an immune response is
detected, e.g.,
antibodies specific for the antigen are detected in the mouse serum, the mouse
spleen is
harvested and splenocytes isolated. The splenocytes are then fused by well-
known
techniques to any suitable myeloma cells, for example cells from cell line
SP20 available
from the ATCC. Hybridomas are selected and cloned by limited dilution. The
hybridoma
20 clones are then assayed by methods known in the art for cells that secrete
antibodies capable
of binding a polypeptide of the invention. Ascites fluid, which generally
contains high levels
of antibodies, can be generated by immunizing mice with positive hybridoma
clones.
Accordingly, the present invention provides methods of generating monoclonal
antibodies as well as antibodies produced by the method comprising culturing a
hybridoma
25 cell secreting an antibody of the invention wherein, preferably, the
hybridoma is generated by
fusing splenocytes isolated from a mouse immunized with an antigen of the
invention with
myeloma cells and then screening the hybridomas resulting from the fusion for
hybridoma
clones that secrete an antibody able to bind a polypeptide of the invention.
Antibody fragments which recognize specific epitopes may be generated by known
30 techniques. For example, Fab and F(ab')2 fragments of the invention may be
produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain
(to
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66
produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2
fragments contain
the variable region, the light chain constant region and the CH1 domain of the
heavy chain.
For example, the antibodies of the present invention can also be generated
using
various 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 a particular embodiment, such phage
can be
utilized to display antigen-binding domains expressed from a repertoire or
combinatorial
antibody library (e.g., human or murine). Phage expressing an antigen binding
domain that
binds the antigen of interest can be selected or identified with antigen,
e.g., using labeled
antigen or antigen bound or captured to a solid surface or bead. Phage used in
these methods
are typically filamentous phage including fd and M13 binding domains expressed
from phage
with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused
to either the
phage gene III or gene VIII protein. Examples of phage display methods that
can be used to
make the antibodies of the present invention include 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/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; 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.
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 in detail below. For example, 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); and 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).
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67
Examples of techniques which can be used to produce single-chain Fvs and
antibodies
include those described in U.S. Patents 4,946,778 and 5,258,498; Huston et
al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra
et al.,
Science 240:1038-1040 (1988). For some uses, including in vivo use of
antibodies in humans
and in vitro detection assays, it may be preferable to use chimeric,
humanized, or human
antibodies. A chimeric antibody is a molecule in which different portions of
the antibody are
derived from different animal species, such as antibodies having a variable
region derived
from a murine monoclonal antibody and a 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); Gillies et al., ( 1989) J.
Immunol. Methods
125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are
incorporated
herein by reference in their entirety. Humanized antibodies are antibody
molecules from non-
human species antibody that binds the desired antigen having one or more
complementarity
determining regions (CDRs) from the non-human species and a framework region
from a
human immunoglobulin molecule. Often, framework residues in the human
framework
regions will be substituted with the corresponding residue from the CDR donor
antibody to
alter, preferably improve, antigen binding. These 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.) Antibodies
can be humanized
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).
Completely human antibodies are particularly desirable for therapeutic
treatment of
human patients. Human antibodies can be made by a variety of methods known in
the art
including phage display methods described above using antibody libraries
derived from
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68
human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and
4,716,111; and
PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein
by
reference in its entirety.
Human antibodies can also be produced using transgenic mice which are
incapable of
expressing functional endogenous immunoglobulins, but which can express human
immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene
complexes may be introduced randomly or by homologous recombination into mouse
embryonic stem cells. Alternatively, the human variable region, constant
region, and
diversity region may be introduced into mouse embryonic stem cells in addition
to the human
heavy and light chain genes. The mouse heavy and light chain immunoglobulin
genes may
be rendered non-functional separately or simultaneously with the introduction
of human
immunoglobulin loci by homologous recombination. In particular, homozygous
deletion of
the JH region prevents endogenous antibody production. The modified embryonic
stem cells
are expanded and microinjected into blastocysts to produce chimeric mice. The
chimeric
mice are then bred to produce homozygous offspring which express human
antibodies. The
transgenic mice are immunized in the normal fashion with a selected antigen,
e.g., all or a
portion of a polypeptide of the invention. Monoclonal antibodies directed
against the antigen
can be obtained from the immunized, transgenic mice using conventional
hybridoma
technology. The human immunoglobulin transgenes harbored by the transgenic
mice
rearrange during B cell differentiation, and subsequently undergo class
switching and somatic
mutation. Thus, using such a technique, it is possible to produce
therapeutically useful IgG,
IgA, IgM and IgE antibodies. For an overview of this technology for producing
human
antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a
detailed
discussion of this technology 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, which are incorporated by
reference herein
in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA)
and
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Genpharm (San Jose, CA) can be engaged to provide human antibodies directed
against a
selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be
generated
using a technique referred to as "guided selection." In this approach a
selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of
a completely
human antibody recognizing the same epitope. (Jespers et al., Biotechnology
12:899-903
( 1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be
utilized to
generate anti-idiotype antibodies that "mimic" polypeptides of the invention
using techniques
well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.
7(5):437-
444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,
antibodies
which bind to and competitively inhibit polypeptide multimerization and/or
binding of a
polypeptide of the invention to a ligand can be used to generate anti-
idiotypes that "mimic"
the polypeptide multimerization and/or binding domain and, as a consequence,
bind to and
neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of
such anti-idiotypes can be used in therapeutic regimens to neutralize
polypeptide ligand. For
example, such anti-idiotypic antibodies can be used to bind a polypeptide of
the invention
and/or to bind its ligands/receptors, and thereby block its biological
activity.
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide
sequence
encoding an antibody of the invention and fragments thereof. The invention
also
encompasses polynucleotides that hybridize under stringent or lower stringency
hybridization
conditions, e.g., as defined supra, to polynucleotides that encode an
antibody, preferably, that
specifically binds to a polypeptide of the invention, preferably, an antibody
that binds to a
polypeptide having the amino acid sequence of SEQ ID N0:2.
The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the nucleotide
sequence of the antibody is known, 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
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oligonucleotides containing portions of the sequence encoding the antibody,
annealing and
ligating of those oligonucleotides, and then amplification of the ligated
oligonucleotides by
PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from
nucleic
5 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 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
10 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
15 art.
Once the nucleotide sequence and corresponding amino acid sequence of the
antibody
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
20 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.
25 In a specific embodiment, the amino acid sequence of the heavy and/or light
chain
variable domains may be inspected to identify the sequences of the
complementarity
determining regions (CDRs) by methods that are well known in the art, e.g., by
comparison
to known amino acid sequences of other heavy and light chain variable regions
to determine
the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or
30 more of the CDRs may be inserted within framework regions, e.g., into human
framework
regions to humanize a non-human antibody, as described supra. The framework
regions may
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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). Preferably, the polynucleotide generated by the
combination of the
framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the
S invention. Preferably, as discussed supra, one or more amino acid
substitutions may be made
within the framework regions, and, preferably, the 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 lacking one or more
intrachain
disulfide bonds. Other alterations to the polynucleotide are encompassed by
the present
invention and within the skill of the art.
In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al.,
Nature 312:604-
608 ( 1984); Takeda et al., Nature 314:452-454 ( 1985)) by splicing genes from
a mouse
antibody molecule of appropriate antigen specificity together with genes from
a human
antibody molecule of appropriate biological activity can be used. As described
supra, a
chimeric antibody is a molecule in which different portions are derived from
different animal
species, such as those having a variable region derived from a murine mAb and
a human
immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain
antibodies (U.S.
Patent No. 4,946,778; Bird, Science 242:423- 42 ( 1988); Huston et al., Proc.
Natl. Acad. Sci.
USA 85:5879-5883 ( 1988); and Ward et al., Nature 334:544-54 ( 1989)) can be
adapted to
produce single chain antibodies. Single chain antibodies are formed by linking
the heavy and
light chain fragments of the Fv region via an amino acid bridge, resulting in
a single chain
polypeptide. Techniques for the assembly of functional Fv fragments in E. coli
may also be
used (Skerra et al., Science 242:1038- 1041 ( 1988)).
Methods of Producing Antibodies
The antibodies 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.
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Recombinant expression of an antibody of the invention, or fragment,
derivative or
analog thereof, (e.g., a heavy or light chain of an antibody of the invention
or a single chain
antibody of the invention), requires construction of an expression vector
containing a
polynucleotide that encodes the antibody. Once a polynucleotide encoding an
antibody
S molecule or a heavy or light chain of an antibody, or portion thereof
(preferably containing
the heavy or light chain variable domain), of the invention has been obtained,
the vector 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, in 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, or a heavy or light chain thereof, or a heavy or light chain
variable domain,
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) and the variable
domain of the
antibody may be cloned into such a vector for expression of the entire heavy
or light chain.
The expression vector is 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 a polynucleotide
encoding an
antibody of the invention, or a heavy or light chain thereof, or a single
chain antibody of the
invention, operably linked to a heterologous promoter. In preferred
embodiments for the
expression of double-chained antibodies, 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.
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
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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., Saccharomyces, 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 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)).
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 J.
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 S-transferase (GST).
In general,
such fusion proteins are soluble and can easily be purified from lysed cells
by adsorption and
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74
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.
In an insect system, Autographa cadifornica nuclear polyhedrosis virus (AcNPV)
is
used as a vector to express foreign genes. The virus grows in Spodoptera
frugiperda cells.
The antibody coding sequence 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).
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 in vitro or in vivo recombination. Insertion in a non-
essential region
of the viral genome (e.g., region E1 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 81:355-359 (1984)). 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 Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
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
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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,
MDCK, 293, 3T3, WI38, 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
5 example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. For example, cell lines which stably express the antibody molecule
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
10 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
15 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 express
the antibody
molecule. Such engineered cell lines may be particularly useful in screening
and evaluation
of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the
herpes
20 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 22:817 (
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: dhfr, which confers
resistance to
25 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);
30 Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.
Biochem.
62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which
confers
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76
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.
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 antibody gene, production of the antibody will also increase (Grouse
et al., Mol.
Cell. Biol. 3:257 (1983)).
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 should be 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 77:2197 ( 1980)). The coding sequences for the heavy and
light chains
may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal,
chemically synthesized; or recombinantly expressed, it may be purified by any
method
known in the art for purification of an immunoglobulin molecule, 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. In addition,
the antibodies of
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77
the present invention or fragments thereof can be fused to heterologous
polypeptide
sequences described herein or otherwise known in the art, to facilitate
purification.
The present invention encompasses antibodies recombinantly fused or chemically
conjugated (including both covalent and non-covalent conjugations) to a
polypeptide (or
portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100
amino acids of the
polypeptide) of the present invention to generate fusion proteins. The fusion
does not
necessarily need to be direct, but may occur through linker sequences. The
antibodies may be
specific for antigens other than polypeptides (or portion thereof, preferably
at least 10, 20,
30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the
present invention. For
example, antibodies may be used to target the polypeptides of the present
invention to
particular cell types, either in vitro or in vivo, by fusing or conjugating
the polypeptides of the
present invention to antibodies specific for particular cell surface
receptors. Antibodies fused
or conjugated to the polypeptides of the present invention may also be used in
in vitro
immunoassays and purification methods using methods known in the art. See
e.g., Harbor et
al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al.,
Immunol. Lett.
39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432
(1992); Fell et
al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in
their entireties.
The present invention further includes compositions comprising the
polypeptides of
the present invention fused or conjugated to antibody domains other than the
variable regions.
For example, the polypeptides of the present invention may be fused or
conjugated to an
antibody Fc region, or portion thereof. The antibody portion fused to a
polypeptide of the
present invention may comprise the constant region, hinge region, CH1 domain,
CH2
domain, and CH3 domain or any combination of whole domains or portions
thereof. The
polypeptides may also be fused or conjugated to the above antibody portions to
form
multimers. For example, Fc portions fused to the polypeptides of the present
invention can
form dimers through disulfide bonding between the Fc portions. Higher
multimeric forms
can be made by fusing the polypeptides to portions of IgA and IgM. Methods for
fusing or
conjugating the polypeptides of the present invention 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 91/06570;
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et
al., J.
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78
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).
As discussed, supra, the polypeptides corresponding to a polypeptide,
polypeptide
fragment, or a variant of SEQ ID N0:2 may be fused or conjugated to the above
antibody
portions to increase the in vivo half life of the polypeptides or for use in
immunoassays using
methods known in the art. Further, the polypeptides corresponding to SEQ ID
N0:2 may be
fused or conjugated to the above antibody portions to facilitate purification.
One reported
example describes chimeric proteins 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
mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86
(1988).
The polypeptides of the present invention fused or conjugated to an antibody
having
disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in binding and
neutralizing other molecules, than the monomeric secreted protein or protein
fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc
part in a
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
for example,
improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
the Fc part
after the fusion protein has been expressed, detected, and purified, would be
desired. For
example, the Fc portion may hinder therapy and diagnosis if the fusion protein
is used as an
antigen for immunizations. In drug discovery, for example, human proteins,
such as hIL-5,
have been fused with Fc portions for the purpose of high-throughput screening
assays to
identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition
8:52-58 (1995);
Johanson et al., J. Biol. Chem. 270:9459-9471 ( 1995).
Moreover, the antibodies or fragments thereof of the present invention can be
fused to
marker sequences, such as a peptide to facilitate purification. In preferred
embodiments, the
marker amino acid sequence is a hexa-histidine peptide, 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
"HA" tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein
(Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
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The present invention further encompasses antibodies or fragments thereof
conjugated
to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for
example, monitor 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 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
fragment thereof) 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 horseradish
peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic
group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable
fluorescent materials include 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 ''5I,
"'I, "'In or 9yTc.
Further, an antibody or fragment 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, z'3Bi. A cytotoxin or
cytotoxic agent
includes any agent that is detrimental to cells. Examples include paclitaxol,
cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,
mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs thereof.
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
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(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin
C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents
5 (e.g., vincristine and vinblastine).
The conjugates of the invention 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, for
example, a toxin
10 such as abrin, ricin A, pseudomonas exotoxin, or diphtheria 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 97/33899), AIM II (See, International
Publication
No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574
(1994)), VEGI
15 (See, International Publication No. WO 99/23105), CD40 Ligand, a thrombotic
agent or an
anti- angiogenic agent, e.g., angiostatin, endostatin or VEGI (See,
International Publication
No. WO 99/23105); or, biological response modifiers such as, for example,
lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophage
colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor
("G-CSF"), or
20 other growth factors.
Antibodies 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.
25 Techniques for conjugating such 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,
Inc. 1987);
30 Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et
al. (eds.), pp.
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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).
Alternatively, an antibody can be conjugated to a second antibody to form ari
antibody
heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is
incorporated
herein by reference in its entirety.
An antibody, 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.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of cell
lines
and biological samples. The translation product of the gene of the present
invention may be
useful as a cell specific marker, or more specifically as a cellular marker
that is differentially
expressed at various stages of differentiation and/or maturation of particular
cell types.
Monoclonal antibodies directed against a specific epitope, or combination of
epitopes, will
allow for the screening of cellular populations expressing the marker. Various
techniques can
be utilized using monoclonal antibodies to screen for cellular populations
expressing the
marker(s), 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 5,985,660; and Morrison et al., Cell, 96:737-49 ( 1999)).
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.
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Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding by
any
method known in the art. The immunoassays which can be used, 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, 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).
Immunoprecipitation 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 NaCI, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented
with
protein phosphatase andlor 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., 1-4 hours) at 4~C, adding protein A and/or protein G sepharose beads to
the cell lysate,
incubating for about an hour or more at 4° 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 further discussion regarding
immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular
Biology, Vol. l,
John Wiley & Sons, Inc., New York at 10.16.1.
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
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83
(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., 'ZP or 'z5I) 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. l, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter
plate
with the antigen, adding the antibody of interest conjugated to a detectable
compound such as
an enzymatic substrate (e.g:, horseradish peroxidase or alkaline phosphatase)
to the well and
incubating for a period of time, and detecting the presence of the antigen. 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, a second antibody
conjugated to a
detectable compound may be added following the addition of the antigen of
interest to the
coated well. One of skill in the art would be knowledgeable as to the
parameters that can be
modified to increase the 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. 1, John Wiley & Sons, Inc., New York at
11.2.1.
The binding affinity of an antibody 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., ;H or 'ZSI) 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 interest for a particular antigen 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, the antigen is
incubated with
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antibody of interest conjugated to a labeled compound (e.g., 'H or ''5I) in
the presence of
increasing amounts of an unlabeled second antibody.
Therapeutic Uses
The present invention is further directed to antibody-based therapies which
involve
administering antibodies of the invention to an animal, preferably a mammal,
and most
preferably a human, patient for treating one or more of the disclosed
diseases, disorders, or
conditions. Therapeutic compounds of the invention include, but are not
limited to,
antibodies of the invention (including fragments, analogs and derivatives
thereof as described
herein) and nucleic acids encoding antibodies of the invention (including
fragments, analogs
and derivatives thereof and anti-idiotypic antibodies as described herein).
The antibodies of
the invention can be used to treat, inhibit or prevent diseases, disorders or
conditions
associated with aberrant expression and/or activity of a polypeptide of the
invention,
including, but not limited to, any one or more of the diseases, disorders, or
conditions
described herein (e.g., autoimmune diseases, disorders, or conditions
associated with such
diseases or disorders, including, but not limited to, autoimmune hemolytic
anemia,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis,
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, Guillain-Barre Syndrome,
insulin
dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune
thyroiditis,
hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus,
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 millitus, and adrenergic drug resistance (including adrenergic drug
resistance with
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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, granulamatous,
degenerative, and
atrophic disorders).
5 In a specific embodiment, antibodies of the invention are be used to treat,
inhibit,
prognose, diagnose or prevent rheumatoid arthritis.
In another specific embodiment, antibodies of the invention are used to treat,
inhibit,
prognose, diagnose or prevent systemic lupus erythematosis.
The treatment and/or prevention of diseases, disorders, or conditions
associated with
10 aberrant expression and/or activity of a polypeptide of the invention
includes, but is not
limited to, alleviating symptoms associated with those diseases, disorders or
conditions. The
antibodies of the invention may also be used to target and kill cells
expressing D-SLAM on
their surface and/or cells having D-SLAM bound to their surface. Antibodies of
the invention
may be provided in pharmaceutically acceptable compositions as known in the
art or as
15 described herein.
A summary of the ways in which the antibodies of the present invention may be
used
therapeutically includes binding polynucleotides or polypeptides of the
present invention
locally or systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated
by complement (CDC) or by effector cells (ADCC). Some of these approaches are
described
20 in more detail below. Armed with the teachings provided herein, one of
ordinary skill in the
art will know how to use the antibodies of the present invention for
diagnostic, monitoring or
therapeutic purposes without undue experimentation.
The antibodies of this invention may be advantageously utilized in combination
with
other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic
growth
25 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.
The antibodies of the invention may be administered alone or in combination
with
other types of treatments (e.g., radiation therapy, chemotherapy, hormonal
therapy,
immunotherapy, anti-tumor agents, antibiotics, and immunoglobulin). Generally,
30 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,
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human antibodies, fragments derivatives, analogs, or nucleic acids, are
administered to a
human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing
antibodies against polypeptides or polynucleotides of the present invention,
fragments or
regions thereof, for both immunoassays directed to and therapy of disorders
related to
polynucleotides or polypeptides, including fragments thereof, of the present
invention. Such
antibodies, fragments, or regions, will preferably have an affinity for
polynucleotides or
polypeptides of the invention, including fragments thereof. Preferred binding
affinities
include those with a dissociation constant or Kd less than 5 X 10-Z M, 10-z M,
5 X 10-' M, 10-;
M, 5 X 10-° M, 10-4 M, 5 X 105 M, 10-5 M, 5 X 10-~ M, 10-fi M, 5 X 10-'
M, 10' M, 5 X 10-R M,
108 M, 5 X 10-y M, 10-9 M, 5 X 10-"' M, 10-"' M, 5 X 10-" M, 10-" M, 5 X 10-'z
M, 10-'Z M,~ 5 X
10~" M, 10-'3 M, 5 X 10-'° M, 10-'° M, 5 X 10-'5 M, and 10-'S M.
Gene Therapy
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 a polypeptide
of the invention,
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.
Any of the methods for gene therapy available in the art can be used according
to the
present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
Clinical
Pharmacy 12:488-505 (1993); 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); May, TIBTECH
11(5):155
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).
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In a preferred embodiment, the compound comprises nucleic acid sequences
encoding
an antibody, said nucleic acid sequences being part of expression vectors that
express the
antibody or fragments or chimeric proteins or heavy or light chains thereof in
a suitable host.
In particular, such nucleic acid sequences have 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 a single chain
antibody;
alternatively, the nucleic acid sequences include sequences encoding both the
heavy and light
chains, or fragments thereof, of the antibody.
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.
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
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embodiment, the nucleic acid can be targeted in vivo for cell specific uptake
and expression,
by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO
92/22635;
W092/20316; 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)).
In a specific embodiment, viral vectors that contain nucleic acid sequences
encoding
an antibody of the invention 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 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
6:291-302
(1994), which describes the use of a retroviral vector to deliver the mdrl
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); Kiem 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).
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 W094/12649;
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89
and Wang, et al., Gene Therapy 2:775-783 ( 1995). In a preferred embodiment,
adenovirus
vectors are used.
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).
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.
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 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., Pharmac. 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.
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.
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,
megakaryocytes, granulocytes; various stem or progenitor cells, in particular
hematopoietic
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stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord
blood, peripheral
blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the
patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic
acid
5 sequences encoding an antibody 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 in vitro can
potentially be used
in accordance with this embodiment of the present invention (see e.g. PCT
Publication WO
10 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth.
Cell Bio.
21A:229 ( 1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 ( 1986)).
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
15 appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
The compounds or pharmaceutical compositions of the invention are preferably
tested
in vitro, and then in vivo for the desired therapeutic or prophylactic
activity, prior to use in
20 humans. For example, in vitro assays to demonstrate the therapeutic or
prophylactic utility of
a compound or pharmaceutical composition include, the effect of a compound on
a cell line
or a patient tissue sample. The effect of the compound or composition on the
cell line and/or
tissue sample can be determined utilizing techniques known to those of skill
in the art
including, but not limited to, rosette formation assays and cell lysis assays.
In accordance
25 with the invention, in vitro assays which can be used to determine whether
administration of
a specific compound is indicated, include in vitro cell culture assays in
which a patient tissue
sample is grown in culture, and exposed to or otherwise administered a
compound, and the
effect of such compound upon the tissue sample is observed.
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Therapeutic and/or Prophylactic Administration and Composition
The invention provides methods of treatment, inhibition and prophylaxis by
administration to a subject of an effective amount of a compound or
pharmaceutical
composition of the invention, preferably an antibody of the invention. In a
preferred
embodiment, the compound is substantially purified (e.g., 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 human.
Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional
appropriate formulations and routes of administration can be selected from
among those
described herein below.
Various delivery systems are known and can be used to administer a compound of
the
invention, e.g., encapsulation in liposomes, microparticles, microcapsules,
recombinant cells
capable of expressing the compound, 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 compounds or 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 compounds or
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.
In a specific embodiment, it may be desirable to administer the pharmaceutical
compounds or 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,
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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.
In another embodiment, the compound or composition can be delivered in a
vesicle,
in particular a liposome (see Langer, 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. 317-
327; see
generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a
controlled release system. In one embodiment, a pump may be used (see Langer,
Supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (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, Langer
and Wise
(eds.), CRC Press, 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. 25:351 (1989); Howard et
al.,
J.Neurosurg. 71:105 ( 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)).
Other controlled release systems are discussed in the review by Langer
(Science
249:1527-1533 (1990)).
In a specific embodiment where the compound 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
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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.
The present invention also provides pharmaceutical compositions. Such
compositions
comprise a therapeutically effective amount of a compound, 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 compound,
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.
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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 lignocaine to ease pain at 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 may be mixed prior to administration.
The compounds 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
canons such as those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
The amount of the compound 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 in
vitro or
animal model test systems.
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. Generally, human antibodies have a longer
half-life
within the human body than antibodies from other species due to the immune
response to the
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foreign polypeptides. Thus, lower dosages of human antibodies and less
frequent
administration is often possible. Further, the dosage and frequency of
administration of
antibodies of the invention may be reduced by enhancing uptake and tissue
penetration (e.g.,
into the brain) of the antibodies by modifications such as, for example,
lipidation.
5 The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of
the invention. Optionally associated with such containers) can be a notice in
the form
prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency of
10 manufacture, use or sale for human administration.
Diagnosis and Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically
bind to a
polypeptide of interest can be used for diagnostic purposes to detect,
diagnose, or monitor
15 diseases and/or disorders associated with the aberrant expression and/or
activity of a
polypeptide of the invention. The invention provides for the detection of
aberrant expression
of a polypeptide of interest, comprising (a) assaying the expression of the
polypeptide of
interest in cells or body fluid of an individual using one or more antibodies
specific to the
polypeptide interest and (b) comparing the level of gene expression with a
standard gene
20 expression level, whereby an increase or decrease in the assayed
polypeptide gene expression
level compared to the standard expression level is indicative of aberrant
expression.
The invention provides a diagnostic assay for diagnosing a disorder,
comprising (a)
assaying the expression of the polypeptide of interest in cells or body fluid
of an individual
using one or more antibodies specific to the polypeptide interest and (b)
comparing the level
25 of gene expression with a standard gene expression level, whereby an
increase or decrease in
the assayed polypeptide gene expression level compared to the standard
expression level is
indicative of a particular disorder. With respect to cancer, the presence of a
relatively high
amount of transcript 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
30 appearance of actual clinical symptoms. A more definitive diagnosis of this
type may allow
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health professionals to employ preventative measures or aggressive treatment
earlier thereby
preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a
biological sample
using classical immunohistological methods 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; radioisotopes, such as iodine ('3'I,
'ZSI, "~I, 'z'I),
carbon ('°C), sulfur ('SS), tritium ('H), indium ("SmIn, ";"'In, "'In,
"'In), and technetium (yyTc,
yy"'Tc), thallium (2"'Ti), gallium (~~Ga, ~'Ga), palladium ("'3Pd), molybdenum
('yMo), xenon
('3'Xe) fluorine ('~F) 'S3Sm "'Lu 'S~Gd "yPm '4°La "SYb "'Ho y"Y ~'Sc
'byre 'RRRe '°ZPr
> > > > > > > > > , > > > >
"'SRh, 9'Ru; luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein
and rhodamine, and biotin.
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).
One embodiment of the invention is the detection and diagnosis of a disease or
disorder associated with aberrant expression of a polypeptide of interest 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 molecule which specifically
binds to the
polypeptide of interest; (b) waiting for a time interval following the
administering for
permitting the labeled molecule to preferentially concentrate at sites in the
subject where the
polypeptide is expressed (and for unbound labeled molecule to be cleared to
background
level); (c) determining background level; and (d) detecting the labeled
molecule in the
subject, such that detection of labeled molecule above the background level
indicates that the
subject has a particular disease or disorder associated with aberrant
expression of the
polypeptide of interest. Background level can be determined by various methods
including,
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comparing the amount of labeled molecule detected to a standard value
previously
determined for a particular system. As described herein, specific embodiments
of the
invention are directed to the use of the antibodies of the invention to
quantitate or qualitate
concentrations of cells of B cell lineage or cells of monocytic lineage.
Also as described herein, antibodies of the invention may be used to treat,
diagnose,
or prognose an individual having an immunodeficiency. In a specific
embodiment,
antibodies of the invention are used to treat, diagnose, and/or prognose an
individual having
common variable immunodeficiency disease (CVID) or a subset of this disease.
In another
embodiment, antibodies of the invention are used to diagnose, prognose, treat
or prevent a
disorder characterized by deficient serium immunoglobulin production,
recurrent infections,
and/or immune system dysfunction.
Also as described herein, antibodies of the invention may be used to treat,
diagnose,
or prognose an individual having an autoimmune disease or disorder. In a
specific
embodiment, antibodies of the invention are used to treat, diagnose, and/or
prognose an
individual having systemic lupus erythematosus, or a subset of the disease. In
another
specific embodiment, antibodies of the invention are used to treat, diagnose
and/or prognose
an individual having rheumatoid arthritis, or a subset of this disease.
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 ~ymTc. The labeled antibody
or antibody
fragment 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.,
"Immunopharmacokinetics 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).
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.
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In another embodiment the time interval following administration is 5 to 20
days or 5 to 10
days.
In an embodiment, monitoring of the disease or disorder is carried out by
repeating
the method for diagnosing the disease or disease, for example, one month after
initial
diagnosis, six months after initial diagnosis, one year after initial
diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods
known
in the art for in 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.
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., 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 patent 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).
Kits
The present invention provides kits that can be used in the above methods. In
one
embodiment, a kit comprises an antibody of the invention, preferably a
purified antibody, in
one or more containers. In a specific embodiment, the kits of the present
invention contain a
substantially isolated polypeptide comprising an epitope which is specifically
immunoreactive with an antibody included in the kit. Preferably, the kits of
the present
invention further comprise a control antibody which does not react with the
polypeptide of
interest. In another specific embodiment, the kits of the present invention
comprise two or
more antibodies (monoclonal and/or polyclonal) that recognize the same and/or
different
sequences or regions of the polypeptide of the invention. In another specific
embodiment, the
kits of the present invention contain a means for detecting the binding of an
antibody to a
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polypeptide of interest (e.g., the antibody may be conjugated to a detectable
substrate such as
a fluorescent compound, an enzymatic substrate, a radioactive compound or a
luminescent
compound, or a second antibody which recognizes the first antibody may be
conjugated to a
detectable substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic kit for
use in screening serum containing antibodies specific against proliferative
and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control antibody
that does not
react with the polypeptide of interest. Such a kit may include a substantially
isolated
polypeptide antigen comprising an epitope which is specifically immunoreactive
with at least
one anti-polypeptide antigen antibody. Further, such a kit includes means for
detecting the
binding of said antibody to the antigen (e.g., the antibody may be conjugated
to a fluorescent
compound such as fluorescein or rhodamine which can be detected by flow
cytometry). In
specific embodiments, the kit may include a recombinantly produced or
chemically
synthesized polypeptide antigen. The polypeptide antigen of the kit may also
be attached to a
solid support.
In a more specific embodiment the detecting means of the above-described kit
includes a solid support to which said polypeptide antigen is attached. Such a
kit may also
include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of
the antibody to the polypeptide antigen can be detected by binding of the said
reporter
labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use
in
screening serum containing antigens of the polypeptide of the invention. The
diagnostic kit
includes a substantially isolated antibody specifically immunoreactive with
polypeptide or
polynucleotide antigens, and means for detecting the binding of the
polynucleotide or
polypeptide antigen to the antibody. In one embodiment, the antibody is
attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal antibody.
The
detecting means of the kit may include a second, labeled monoclonal antibody.
Alternatively,
or in addition, the detecting means may include a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase
reagent
having a surface-bound antigen obtained by the methods of the present
invention. After
binding with specific antigen antibody to the reagent and removing unbound
serum
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components by washing, the reagent is reacted with reporter-labeled anti-human
antibody to
bind reporter to the reagent in proportion to the amount of bound anti-antigen
antibody on the
solid support. The reagent is again washed to remove unbound labeled antibody,
and the
amount of reporter associated with the reagent is determined. Typically, the
reporter is an
enzyme which is detected by incubating the solid phase in the presence of a
suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques
for
attaching protein material to solid support material, such as polymeric beads,
dip sticks, 96-
well plate or filter material. These attachment methods generally include non-
specific
adsorption of the protein to the support or covalent attachment of the
protein, typically
through a free amine group, to a chemically reactive group on the solid
support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin
coated plates can
be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this
diagnostic
method. The kit generally includes a support with surface- bound recombinant
antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound anti-antigen
antibody.
The invention further relates to antibodies which act as agonists or
antagonists of the
polypeptides of the present invention. For example, the present invention
includes antibodies
which disrupt the receptor/ligand interactions with the polypeptides of the
invention either
partially or fully. Included are both receptor-specific antibodies and ligand-
specific
antibodies. Included are receptor-specific antibodies which do not prevent
ligand binding but
prevent receptor activation. Receptor activation (i.e., signaling) may be
determined by
techniques described herein or otherwise known in the art. Also included are
receptor-
specific antibodies which both prevent ligand binding and receptor activation.
Likewise,
included are neutralizing antibodies which bind the ligand and prevent binding
of the ligand
to the receptor, as well as antibodies which bind the ligand, thereby
preventing receptor
activation, but do not prevent the ligand from binding the receptor. Further
included are
antibodies which activate the receptor. These antibodies may act as agonists
for either all or
less than all of the biological activities affected by ligand-mediated
receptor activation. The
antibodies may be specified as agonists or antagonists for biological
activities comprising
specific .activities disclosed herein. Further included are antibodies that
bind to D-SLAM
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irrespective of whether D-SLAM is bound to a D-SLAM Receptor. These antibodies
act as
D-SLAM agonists as reflected in an decrease in cellular proliferation in
response to binding
of D-SLAM to a D-SLAM receptor in the presence of these antibodies. The above
antibody
agonists can be made using methods known in the art. See e.g., WO 96/40281; US
Patent
5,811,097; Deng, B. et al., Blood 92(6):1981-1988 (1998); Chen, Z. et al.,
Cancer Res.
58(16):3668-3678 (1998); Harrop, J.A. et al., J. Immunol. 161(4):1786-1794
(1998); Zhu, Z.
et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, D.Y. et al., J. Immunol.
160(7):3170-
3179 (1998); Prat, M. et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard,
V. et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard, J. et al., Cytokinde
9(4):233-241
(1997); Carlson, N.G. et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman, R.E. et
al., Neuron 14(4):755-762 (1995); Muller, Y.A. et al., Structure 6(9):1153-
1167 (1998);
Bartunek, P. et al., Cytokine 8(1):14-20 (1996) (said references incorporated
by reference in
their entireties).
The invention encompasses antibodies that inhibit or reduce the ability of D-
SLAM to
bind D-SLAM receptor in vitro and/or in vivo. In a specific embodiment,
antibodies of the
invention inhibit or reduce the ability of D-SLAM to bind D-SLAM receptor in
vitro. In
another nonexclusive specific embodiment, antibodies of the invention inhibit
or reduce the
ability of D-SLAM to bind D-SLAM receptor in vivo. Such inhibition can be
assayed using
techniques described herein or otherwise known in the art.
The invention also encompasses, antibodies that bind specifically to D-SLAM,
but do
not inhibit the ability of D-SLAM to bind D-SLAM receptor in vitro and/or in
vivo. In a
specific embodiment, antibodies of the invention do not inhibit or reduce the
ability of D
SLAM to bind D-SLAM receptor in vitro. In another nonexclusive specific
embodiment,
antibodies of the invention do not inhibit or reduce the ability of D-SLAM to
bind D-SLAM
receptor in vivo.
As described above, the invention encompasses antibodies that inhibit or
reduce a D-
SLAM-mediated biological activity in vitro and/or in vivo. In a specific
embodiment,
antibodies of the invention inhibit or reduce D-SLAM-mediated inhibition of B
cell
proliferation in vitro. Such inhibition can be assayed by routinely modifying
B cell
proliferation assays described herein or otherwise known in the art. In
another nonexclusive
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specific embodiment, antibodies of the invention inhibit or reduce D-SLAM-
mediated
inhibition of B cell proliferation in vivo.
Alternatively, the invention also encompasses, antibodies that bind
specifically to D
SLAM, but do not inhibit or reduce a D-SLAM-mediated biological activity in
vitro and/or in
vivo (e.g., inhibition of B cell proliferation). In a specific embodiment,
antibodies of the
invention do not inhibit or reduce a D-SLAM-mediated biological activity in
vitro. In
another non-exclusive embodiment, antibodies of the invention do not inhibit
or reduce a D-
SLAM-mediated biological activity in vivo.
As described above, the invention encompasses antibodies that specifically
bind to the
same epitope as at least one of the antibodies specifically referred to
herein, in vitro and/or in
vivo.
The antibodies of the invention also have uses as therapeutics and/or
prophylactics
which include, but are not limited to, in activating monocytes or blocking
monocyte
activation and/or killing specific cell types that express the membrane bound
form of D-
SLAM on their cell surfaces (e.g., to treat, prevent, and/or diagnose
leukemias, lymphomas,
rheumatoid arthritis, and other diseases or conditions associated with these
cell types). In a
specific embodiment, the antibodies of the invention fix complement. In other
specific
embodiments, as further described herein, the antibodies of the invention (or
fragments
thereof) are associated with heterologous polypeptides or nucleic acids (e.g.
toxins, such as,
compounds that bind and activate endogenous cytotoxic effecter systems, and
radioisotopes;
and cytotoxic prodrugs).
As discussed above, antibodies to the D-SLAM polypeptides of the invention
can, in
turn, be utilized to generate anti-idiotype antibodies that "mimic" the D-
SLAM, using
techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J.
7(5):437-444 (1989), and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). Such
neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be
used in therapeutic
regimens to neutralize D-SLAM ligand. For example, such anti-idiotypic
antibodies can be
used to bind D-SLAM, or to bind D-SLAM receptors on the surface of cells of B
cell lineage,
and thereby block D-SLAM-mediated inhibition of B cell activation,
proliferation, and/or
differentiation.
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Fusion Proteins
Any D-SLAM polypeptide can be used to generate fusion proteins. For example,
the
D-SLAM polypeptide, when fused to a second protein, can be used as an
antigenic tag.
Antibodies raised against the D-SLAM polypeptide can be used to indirectly
detect the
second protein by binding to the D-SLAM. Moreover, because secreted proteins
target
cellular locations based on trafficking signals, the D-SLAM polypeptides can
be used as a
targeting molecule once fused to other proteins.
Examples of domains that can be fused to D-SLAM polypeptides include not only
heterologous signal sequences, but also other heterologous functional regions.
The fusion
does not necessarily need to be direct, but may occur through linker
sequences.
In certain preferred embodiments, D-SLAM proteins of the invention comprise,
or
alternatively consist of, fusion proteins wherein the D-SLAM polypeptides are
those
described above as m-n. In preferred embodiments, the application is directed
to nucleic acid
molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to
the
IS nucleic acid sequences encoding polypeptides having the amino acid sequence
of the specific
N- and C-terminal deletions recited herein.
Moreover, fusion proteins may also be engineered to improve characteristics of
the D-
SLAM polypeptide. For instance, a region of additional amino acids,
particularly charged
amino acids, may be added to the N-terminus of the D-SLAM polypeptide to
improve
stability and persistence during purification from the host cell or subsequent
handling and
storage. Also, peptide moieties may be added to the D-SLAM polypeptide to
facilitate
purification. Such regions may be removed prior to final preparation of the D-
SLAM
polypeptide. The addition of peptide moieties to facilitate handling of
polypeptides are
familiar and routine techniques in the art.
Moreover, D-SLAM polypeptides of the present invention, including fragments
thereof, and specifically epitope-bearing fragments may be fused with
heterologous
polypeptide sequences. For example, the polypeptides of the present invention
may be fused
with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or
portions
thereof (CH1, CH2, CH3, and any combination thereof, including both entire
domains and
portions thereof), or albumin (including but not limited to recombinant
albumin), resulting in
chimeric polypeptides. These fusion proteins facilitate purification and show
an increased
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half-life in vivo. One reported example describes chimeric proteins 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 mammalian immunoglobulins. (EP A 394,827; Traunecker
et al.,
Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric
structures (due to
the IgG) can also be more efficient in binding and neutralizing other
molecules, than the
monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J.
Biochem.
270:3958-3964 (1995).) Polynucleotides comprising or alternatively consisting
of nucleic
acids which encode these fusion proteins are also encompassed by the
invention.
Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion
proteins
comprising various portions of constant region of immunoglobulin molecules
together with
another human protein or part thereof. In many cases, the Fc part in a fusion
protein is
beneficial in therapy and diagnosis, and thus can result in, for example,
improved
pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc
part after the
fusion protein has been expressed, detected, and purified, would be desired.
For example, the
Fc portion may hinder therapy and diagnosis if the fusion protein is used as
an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL,-5,
have been
fused with Fc portions for the purpose of high-throughput screening assays to
identify
antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-
58 ( 1995); K.
Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)
Moreover, the D-SLAM polypeptides can be fused to marker sequences, such as a
peptide which facilitates purification of D-SLAM. In preferred embodiments,
the marker
amino acid sequence is a hexa-histidine peptide, 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. Another peptide tag useful for purification, the "HA" tag,
corresponds to an epitope
derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767
(1984).)
Thus, any of these above fusions can be engineered using the D-SLAM
polynucleotides or the polypeptides.
Recombinant and Synthetic Production of D-SLAM
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The present invention also relates to vectors containing the isolated D-SLAM
DNA
molecules of the present invention, host cells which are genetically
engineered with the
recombinant vectors, and the production of polypeptides or fragments thereof
by recombinant
and synthetic techniques. The vector may be, for example, a phage, plasmid,
viral, or
retroviral vector. Retroviral vectors may be replication competent or
replication defective. In
the latter case, viral propagation generally will occur only in complementing
host cells.
D-SLAM polynucleotides may be joined to a vector containing a selectable
marker
for propagation in a host. Generally, a plasmid vector is introduced in a
precipitate, such as a
calcium phosphate precipitate, or in a complex with a charged lipid. If the
vector is a virus, it
may be packaged in vitro using an appropriate packaging cell line and then
transduced into
host cells.
The D-SLAM polynucleotide insert should be operatively linked to an
appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and
tac
promoters, the SV40 early and late promoters and promoters of retroviral LTRs,
to name a
few. Other suitable promoters will be known to the skilled artisan. The
expression
constructs will further contain sites for transcription initiation,
termination, and, in the
transcribed region, a ribosome binding site for translation. The coding
portion of the
transcripts expressed by the constructs will preferably include a translation
initiating codon at
the beginning and a termination codon (UAA, UGA or UAG) appropriately
positioned at the
end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable
marker. Such markers include dihydrofolate reductase, 6418 or neomycin
resistance for
eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance
genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include,
but are not limited to, bacterial cells, such as E. coli, Streptomyces and
Salmonella
typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces
cerevisiae or Pichia
pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and
Spodoptera
Sf9 cells; animal cells such as CI-IO, COS, 293, and Bowes melanoma cells; and
plant cells.
Appropriate culture mediums and conditions for the above-described host cells
are known in
the art.
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Among vectors preferred for use in bacteria include pHE4-5 (ATCC Accession No.
209311; and variations thereof), pQE70, pQE60 and pQE-9, available from
QIAGEN, Inc.;
pBluescript vectors, Phagescript vectors, pNHBA, pNHl6a, pNHl8A, pNH46A,
available
from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRITS
available from Pharmacia Biotech, Inc. Preferred expression vectors for use in
yeast systems
include, but are not limited to, pYES2, pYDI, pTEFI/Zeo, pYES2/GS, pPICZ,
pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1, pPIC3.5K, pPIC9K, and PA0815
(all
available from Invitrogen, Carlsbad, CA). Among preferred eukaryotic vectors
are
pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for
use in
yeast systems include, but are not limited to pYES2, pYDI, pTEFI/Zeo,
pYES2/GS, pPICZ,
pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1, pPIC3.5K, pPIC9K, and
PA0815
(all available from Invitrogen, Carlsbad, CA). Other suitable vectors will be
readily apparent
to the skilled artisan. Other suitable vectors will be readily apparent to the
skilled artisan.
In one embodiment, the yeast Pichia pa.storis is used to express D-SLAM
protein in a
eukaryotic system. Pichia pastoris is a methylotrophic yeast which can
metabolize methanol
as its sole carbon source. A main step in the methanol metabolization pathway
is the
oxidation of methanol to formaldehyde using O~. This reaction is catalyzed by
the enzyme
alcohol oxidase. In order to metabolize methanol as its sole carbon source,
Pichia pastoris
must generate high levels of alcohol oxidase due, in part, to the relatively
low affinity of
alcohol oxidase for OZ. Consequently, in a growth medium depending on methanol
as a main
carbon source, the promoter region of one of the two alcohol oxidise genes
(AOXI ) is highly
active. In the presence of methanol, alcohol oxidise produced from the ADXl
gene
comprises up to approximately 30°10 of the total soluble protein in
Pichia pastoris. See, Ellis,
S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast
5:167-77 (1989);
Tschopp, J.F., et al., Nucl. Acids Re.s. 15:3859-76 (1987). Thus, a
heterologous coding
sequence, such as, for example, a D-SLAM polynucleotide of the present
invention, under the
transcriptional regulation of all or part of the AOXI regulatory sequence is
expressed at
exceptionally high levels in Pichia yeast grown in the presence of methanol.
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In one example, the plasmid vector pPIC9K is used to express DNA encoding a D-
SLAM polypeptide of the invention, as set forth herein, in a Pichea yeast
system essentially
as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins
and J.
Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows
expression
and secretion of a D-SLAM protein of the invention by virtue of the strong
AOXl promoter
linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal
peptide (i.e., leader)
located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD
1,
pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S
1,
pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as
long as the
proposed expression construct provides appropriately located signals for
transcription,
translation, secretion (if desired), and the like, including an in-frame AUG
as required.
In one embodiment, high-level expression of a heterologous coding sequence,
such as,
for example, a D-SLAM polynucleotide of the present invention, may be achieved
by cloning
the heterologous polynucleotide of the invention into an expression vector
such as, for
example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of
methanol.
Introduction of the construct into the host cell can be effected by calcium
phosphate
transfection, DEAE-dextran mediated transfection, cationic lipid-mediated
transfection,
electroporation, transduction, infection, or other methods. Such methods are
described in
many standard laboratory manuals, such as Davis et al., Basic Methods In
Molecular Biology
( 1986). It is specifically contemplated that D-SLAM polypeptides may in fact
be expressed
by a host cell lacking a recombinant vector.
D-SLAM polypeptides can be recovered and purified from recombinant cell
cultures
by well-known methods including ammonium sulfate or ethanol precipitation,
acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography and lectin chromatography. Most preferably, high performance
liquid
chromatography ("HPLC") is employed for purification.
D-SLAM polypeptides, and preferably the secreted form, can also be recovered
from:
products purified from natural sources, including bodily fluids, tissues and
cells, whether
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directly isolated or cultured; 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 D-SLAM polypeptides
may be
glycosylated or may be non-glycosylated. In addition, D-SLAM polypeptides may
also
include an initial modified methionine residue, in some cases as a result of
host-mediated
processes. Thus, it is well known in the art that the N-terminal methionine
encoded by the
translation initiation codon generally is removed with high efficiency from
any protein after
translation in all eukaryotic cells. While the N-terminal methionine on most
proteins also is
efficiently removed in most prokaryotes, for some proteins, this prokaryotic
removal process
is inefficient, depending on the nature of the amino acid to which the N-
terminal methionine
is covalently linked.
In addition to encompassing host cells containing the vector constructs
discussed
herein, the invention also encompasses primary, secondary, and immortalized
host cells of
vertebrate origin, particularly mammalian origin, that have been engineered to
delete or
replace endogenous genetic material (e.g., D-SLAM coding sequence), and/or to
include
genetic material (e.g., heterologous polynucleotide sequences) that is
operably associated
with D-SLAM polynucleotides of the invention, and which activates, alters,
and/or amplifies
endogenous D-SLAM polynucleotides. For example, techniques known in the art
may be
used to operably associate heterologous control regions (e.g., promoter and/or
enhancer) and
endogenous D-SLAM polynucleotide sequences via homologous recombination (see,
e.g.,
U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No.
WO
96/29411, published September 26, 1996; International Publication No. WO
94/12650,
published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-
8935 (1989);
and Zijlstra et al., Nature 342:435-438 ( 1989), the disclosures of each of
which are
incorporated by reference in their entireties).
In addition, polypeptides of the invention can be chemically synthesized using
techniques known in the art (e~, 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 the D-SLAM
polypeptides of
the invention can be synthesized by use of a peptide synthesizer. Furthermore,
if desired,
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nonclassical amino acids or chemical amino acid analogs can be introduced as a
substitution
or addition into the D-SLAM polynucleotide sequence. Non-classical amino acids
include,
but are not limited 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).
The invention encompasses D-SLAM polypeptides 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.
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,
isotopic or affinity label to allow for detection and isolation of the
protein.
Also provided by the invention are chemically modified derivatives of D-SLAM
which may provide additional advantages such as increased solubility,
stability and
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
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modified at random positions within the molecule, or at predetermined
positions within the
molecule and may include one, two, three or more attached chemical moieties.
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~or lack of antigenicity and other known effects of the
polyethylene
glycol to a therapeutic protein or analog).
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 protein.
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 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 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.
One may specifically desire proteins chemically modified at the N-terminus.
Using
polyethylene glycol as an illustration of the present composition, 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
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protein molecules. Selective proteins chemically modified at the N-terminus
modification
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 protein. Under the appropriate reaction
conditions, substantially
selective derivatization of the protein at the N-terminus with a carbonyl
group containing
polymer is achieved.
The D-SLAM polypeptides of the invention may be in monomers or multimers
(i.e.,
dimers, trimers, tetramers and higher multimers). Accordingly, the present
invention relates
to monomers and multimers of the D-SLAM polypeptides of the invention, their
preparation,
and compositions (preferably, pharmaceutical compositions) containing them. In
specific
embodiments, the polypeptides of the invention are monomers, dimers, trimers
or tetramers.
In additional embodiments, the multimers of the invention are at least dimers,
at least trimers,
or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used
herein, the term homomer, refers to a multimer containing only D-SLAM
polypeptides of the
invention (including D-SLAM fragments, variants, splice variants, and fusion
proteins, as
described herein). These homomers may contain D-SLAM polypeptides having
identical or
different amino acid sequences. In a specific embodiment, a homomer of the
invention is a
multimer containing only D-SLAM polypeptides having an identical amino acid
sequence.
In another specific embodiment, a homomer of the invention is a multimer
containing D-
SLAM polypeptides having different amino acid sequences. In specific
embodiments, the
multimer of the invention is a homodimer (e.g., containing D-SLAM polypeptides
having
identical or different amino acid sequences) or a homotrimer (e.g., containing
D-SLAM
polypeptides having identical and/or different amino acid sequences). In
additional
embodiments, the homomeric multimer of the invention is at least a homodimer,
at least a
homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more
heterologous polypeptides (i.e., polypeptides of different proteins) in
addition to the D-
SLAM polypeptides of the invention. In a specific embodiment, the multimer of
the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional
embodiments,
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the homomeric multimer of the invention is at least a homodimer, at least a
homotrimer, or at
least a homotetramer.
Multimers 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, multimers of the invention, such as, for
example,
homodimers or homotrimers, are formed when polypeptides of the invention
contact one
another in solution. In another embodiment, heteromultimers of the invention,
such as, for
example, heterotrimers or 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, multimers of the invention are formed by covalent associations
with and/or
between the D-SLAM 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 ID N0:2, or contained in the polypeptide encoded by the clone HDPJ039). 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 D-SLAM fusion
protein. In
one example, covalent associations are between the heterologous sequence
contained in a
fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a
specific
example, the covalent associations are between the heterologous sequence
contained in a D-
SLAM-Fc fusion protein of the invention (as described herein). In another
specific example,
covalent associations of fusion proteins of the invention are between
heterologous
polypeptide sequence from another Secreted Lymphocyte Activation Molecule
(SLAM)
family 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
embodiment, two or more polypeptides of the invention are joined through
peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No. 5,073,627
(hereby
incorporated by reference). Proteins comprising multiple polypeptides of the
invention
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separated by peptide linkers may be produced using conventional recombinant
DNA
technology.
Another method for preparing multimer polypeptides of the invention involves
use of
polypeptides of the invention fused to a leucine zipper or isoleucine zipper
polypeptide
sequence. Leucine zipper and isoleucine zipper domains are polypeptides that
promote
multimerization of the proteins in which they are found. Leucine zippers were
originally
identified in several DNA-binding proteins (Landschulz et al., Science
240:1759, (1988)),
and have since been found in a variety of different proteins. Among the known
leucine
zippers are naturally occurring peptides and derivatives thereof that dimerize
or trimerize.
Examples of leucine zipper domains suitable for producing soluble multimeric
proteins of
the invention are those described in PCT application WO 94/10308, hereby
incorporated by
reference. Recombinant fusion proteins comprising a polypeptide of the
invention fused to a
polypeptide sequence that dimerizes or trimerizes in solution are expressed in
suitable host
cells, and the resulting soluble multimeric fusion protein is recovered from
the culture
supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced
biological activity. Preferred leucine zipper moieties and isoleucine moieties
are those that
preferentially form trimers. One example is a leucine zipper derived from lung
surfactant
protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994))
and in U.S.
patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides
derived from naturally occurring trimeric proteins may be employed in
preparing trimeric
polypeptides of the invention.
In another example, proteins of the invention are associated by interactions
between
Flag~ polypeptide sequence contained in fusion proteins of the invention
containing Flag~
polypeptide seuqence. In a further embodiment, associations proteins of the
invention are
associated by interactions between heterologous polypeptide sequence contained
in Flag~
fusion proteins of the invention and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical techniques
known in
the art. For example, polypeptides desired to be contained in the multimers of
the invention
may be chemically cross-linked using linker molecules and linker molecule
length
optimization techniques known in the art (see, e.g., US Patent Number
5,478,925, which is
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herein incorporated by reference in its entirety). Additionally, multimers of
the invention
may be generated using techniques known in the art to form one or more inter-
molecule
cross-links between the cysteine residues located within the sequence of the
polypeptides
desired to be contained in the multimer (see, e.g., US Patent Number
5,478,925, which is
herein incorporated by reference in its entirety). Further, polypeptides of
the invention may
be routinely modified by the addition of cysteine or biotin to the C terminus
or N-terminus of
the polypeptide and techniques known in the art may be applied to generate
multimers
containing one or more of these modified polypeptides (see, e.g., US Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques
known in the art may be applied to generate liposomes containing the
polypeptide
components desired to be contained in the multimer of the invention (see,
e.g., US Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering
techniques known in the art. In one embodiment, polypeptides contained in
multimers of the
invention are produced recombinantly using fusion protein technology described
herein or
otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety). In a specific embodiment,
polynucleotides coding
for a homodimer of the invention are generated by ligating a polynucleotide
sequence
encoding a polypeptide of the invention to a sequence encoding a linker
polypeptide and then
further to a synthetic polynucleotide encoding the translated product of the
polypeptide in the
reverse orientation from the original C-terminus to the N-terminus (lacking
the leader
sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by reference
in its entirety). In another embodiment, recombinant techniques described
herein or
otherwise known in the art are applied to generate recombinant polypeptides of
the invention
which contain a transmembrane domain (or hyrophobic or signal peptide) and
which can be
incorporated by membrane reconstitution techniques into liposomes (see, e.g.,
US Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
Uses of the D-SLAM Polynucleotides
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The D-SLAM polynucleotides identified herein can be used in numerous ways as
reagents. The following description should be considered exemplary and
utilizes known
techniques.
There exists an ongoing need to identify new chromosome markers, since few
chromosome marking reagents, based on actual sequence data (repeat
polymorphisms), are
presently available.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the sequences shown in SEQ ID NO:I. Primers can be
selected
using computer analysis so that primers do not span more than one predicted
exon in the
genomic DNA. These primers are then used for PCR screening of somatic cell
hybrids
containing individual human chromosomes. Only those hybrids containing the
human D-
SLAM gene corresponding to the SEQ ID NO:1 will yield an amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per day
using a single thermal cycler. Moreover, sublocalization of the D-SLAM
polynucleotides can
be achieved with panels of specific chromosome fragments. Other gene mapping
strategies
that can be used include in situ hybridization, prescreening with labeled flow-
sorted
chromosomes, and preselection by hybridization to construct chromosome
specific-cDNA
libraries.
Precise chromosomal location of the D-SLAM polynucleotides can also be
achieved
using fluorescence in situ hybridization (FISH) of a metaphase chromosomal
spread. This
technique uses polynucleotides as short as 500 or 600 bases; however,
polynucleotides 2,000-
4,000 by are preferred. For a review of this technique, see Verma et al.,
"Human
Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).
For chromosome mapping, the D-SLAM polynucleotides can be used individually
(to
mark a single chromosome or a single site on that chromosome) or in panels
(for marking
multiple sites and/or multiple chromosomes). Preferred polynucleotides
correspond to the
noncoding regions of the cDNAs because the coding sequences are more likely
conserved
within gene families, thus increasing the chance of cross hybridization during
chromosomal
mapping.
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Once a polynucleotide has been mapped to a precise chromosomal location, the
physical position of the polynucleotide can be used in linkage analysis.
Linkage analysis
establishes coinheritance between a chromosomal location and presentation of a
particular
disease. (Disease mapping data are found, for example, in V. McKusick,
Mendelian
Inheritance in Man (available on line through Johns Hopkins University Welch
Medical
Library) .) Assuming 1 megabase mapping resolution and one gene per 20 kb, a
cDNA
precisely localized to a chromosomal region associated with the disease could
be one of 50-
500 potential causative genes.
Thus, once coinheritance is established, differences in the D-SLAM
polynucleotide
and the corresponding gene between affected and unaffected individuals can be
examined.
First, visible structural alterations in the chromosomes, such as deletions or
translocations,
are examined in chromosome spreads or by PCR. If no structural alterations
exist, the
presence of point mutations are ascertained. Mutations observed in some or all
affected
individuals, but not in normal individuals, indicates that the mutation may
cause the disease.
However, complete sequencing of the D-SLAM polypeptide and the corresponding
gene
from several normal individuals is required to distinguish the mutation from a
polymorphism.
If a new polymorphism is identified, this polymorphic polypeptide can be used
for further
linkage analysis.
Furthermore, increased or decreased expression of the gene in affected
individuals as
compared to unaffected individuals can be assessed using D-SLAM
polynucleotides. Any of
these alterations (altered expression, chromosomal rearrangement, or mutation)
can be used
as a diagnostic or prognostic marker.
In addition to the foregoing, a D-SLAM polynucleotide can be used to control
gene
expression through triple helix formation or antisense DNA or RNA. Both
methods rely on
binding of the polynucleotide to DNA or RNA. For these techniques, preferred
polynucleotides are usually 20 to 40 bases in length and complementary to
either the region
of the gene involved in transcription (triple helix - see Lee et al., Nucl.
Acids Res. 6:3073
( 1979); Cooney et al., Science 241:456 ( 1988); and Dervan et al., Science
251:1360 ( 1991 ) )
or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560 (1991);
Oligodeoxy-
nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton,
FL ( 1988).)
Triple helix formation optimally results in a shut-off of RNA transcription
from DNA, while
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antisense RNA hybridization blocks translation of an mRNA molecule into
polypeptide.
Both techniques are effective in model systems, and the information disclosed
herein can be
used to design antisense or triple helix polynucleotides in an effort to
treat, diagnose, detect,
and/or prevent disease.
D-SLAM polynucleotides are also useful in gene therapy. One goal of gene
therapy is
to insert a normal gene into an organism having a defective gene, in an effort
to correct the
genetic defect. D-SLAM offers a means of targeting such genetic defects in a
highly accurate
manner. Another goal is to insert a new gene that was not present in the host
genome,
thereby producing a new trait in the host cell.
The D-SLAM polynucleotides are also useful for identifying individuals from
minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel. In this
technique, an individual's genomic DNA is digested with one or more
restriction enzymes,
and probed on a Southern blot to yield unique bands for identifying personnel.
This method
does not suffer from the current limitations of "Dog Tags" which can be lost,
switched, or
stolen, making positive identification difficult. The D-SLAM polynucleotides
can be used as
additional DNA markers for RFLP.
The D-SLAM polynucleotides can also be used as an alternative to RFLP, by
determining the actual base-by-base DNA sequence of selected portions of an
individual's
genome. These sequences can be used to prepare PCR primers for amplifying and
isolating
such selected DNA, which can then be sequenced. Using this technique,
individuals can be
identified because each individual will have a unique set of DNA sequences.
Once an unique
ID database is established for an individual, positive identification of that
individual, living or
dead, can be made from extremely small tissue samples.
Forensic biology also benefits from using DNA-based identification techniques
as
disclosed herein. DNA sequences taken from very small biological samples such
as tissues,
e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be
amplified using PCR.
In one prior art technique, gene sequences amplified from polymorphic loci,
such as DQa
class II HLA gene, are used in forensic biology to identify individuals.
(Erlich, H., PCR
Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are
amplified,
they are digested with one or more restriction enzymes, yielding an
identifying set of bands
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on a Southern blot probed with DNA corresponding to the DQa class II HLA gene.
Similarly, D-SLAM polynucleotides can be used as polymorphic markers for
forensic
purposes.
There is also a need for reagents capable of identifying the source of a
particular
tissue. Such need arises, for example, in forensics when presented with tissue
of unknown
origin. Appropriate reagents can comprise, or alternatively consist of, for
example, DNA
probes or primers specific to particular tissue prepared from D-SLAM
sequences. Panels of
such reagents can identify tissue by species and/or by organ type. In a
similar fashion, these
reagents can be used to screen tissue cultures for contamination.
Because D-SLAM is found expressed in dendritic cells, T cell lymphoma, lymph
node, spleen, thymus, small intestine, and uterus, D-SLAM polynucleotides are
useful as
hybridization probes for differential identification of the tissues) or cell
types) present in a
biological sample. Similarly, polypeptides and antibodies directed to D-SLAM
polypeptides
are useful to provide immunological probes for differential identification of
the tissues) or
cell type(s). In addition, for a number of disorders of the above tissues or
cells, particularly
of the immune system, significantly higher or lower levels of D-SLAM gene
expression may
be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily
fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual
having such a
disorder, relative to a "standard" D-SLAM gene expression level, i.e., the D-
SLAM
expression level in healthy tissue from an individual not having the immune
system disorder.
Thus, the invention provides a diagnostic method of a disorder, which
involves: (a)
assaying D-SLAM gene expression level in cells or body fluid of an individual;
(b)
comparing the D-SLAM gene expression level with a standard D-SLAM gene
expression
level, whereby an increase or decrease in the assayed D-SLAM gene expression
level
compared to the standard expression level is indicative of disorder in the
immune system.
In the very least, the D-SLAM polynucleotides can be used as molecular weight
markers on Southern gels, as diagnostic probes for the presence of a specific
mRNA in a
particular cell type, as a probe to "subtract-out" known sequences in the
process of
discovering novel polynucleotides, for selecting and making oligomers for
attachment to a
"gene chip" or other support, to raise anti-DNA antibodies using DNA
immunization
techniques, and as an antigen to elicit an immune response.
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Uses of D-SLAM Polypentides
D-SLAM polypeptides can be used in numerous ways. The following description
should be considered exemplary and utilizes known techniques.
D-SLAM polypeptides can be used to assay protein levels in a biological sample
using antibody-based techniques. For example, protein expression in tissues
can be studied
with classical immunohistological methods. (Jalkanen, M., et al., J. Cell.
Biol. 101:976-985
(1985); Jalkanen, M., 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, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium
(3H), indium ( 1 l2In), and technetium (99mTc), and fluorescent labels, such
as fluorescein
and rhodamine, and biotin.
In addition to assaying secreted protein levels in a biological sample,
proteins can also
be detected in vivo by imaging. Antibody labels or markers for in vivo imaging
of protein
include those detectable by X-radiography, NMR or ESR. For X-radiography,
suitable labels
include radioisotopes such as barium or cesium, which emit detectable
radiation but are not
overtly harmful to the subject. Suitable markers for NMR and ESR include those
with a
detectable characteristic spin, such as deuterium, which may be incorporated
into the
antibody by labeling of nutrients for the relevant hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with
an
appropriate detectable imaging moiety, such as a radioisotope (for example,
131I, 112In,
99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic
resonance, is
introduced (for example, parenterally, subcutaneously, or intraperitoneally)
into the mammal.
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 99mTc. The labeled antibody
or antibody
fragment 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.,
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"Immunopharmacokinetics 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).)
Thus, the invention provides a diagnostic method of a disorder, which involves
(a)
assaying the expression of D-SLAM polypeptide in cells or body fluid of an
individual; (b)
comparing the level of gene expression with a standard gene expression level,
whereby an
increase or decrease in the assayed D-SLAM polypeptide gene expression level
compared to
the standard expression level is indicative of a disorder.
Moreover, D-SLAM polypeptides can be used to treat, diagnose, detect, and/or
prevent disease. For example, patients can be administered D-SLAM polypeptides
in an
effort to replace absent or decreased levels of the D-SLAM polypeptide (e.g.,
insulin), to
supplement absent or decreased levels of a different polypeptide (e.g.,
hemoglobin S for
hemoglobin B), to inhibit the activity of a polypeptide (e.g., an oncogene),
to activate the
activity of a polypeptide (e.g., by binding to a receptor), to reduce the
activity of a membrane
bound receptor by competing with it for free ligand (e.g., soluble TNF
receptors used in
reducing inflammation), or to bring about a desired response (e.g., blood
vessel growth).
Similarly, antibodies directed to D-SLAM polypeptides can also be used to
treat,
diagnose, detect, and/or prevent disease. For example, administration of an
antibody directed
to a D-SLAM polypeptide can bind and reduce overproduction of the polypeptide.
Similarly,
administration of an antibody can activate the polypeptide, such as by binding
to a
polypeptide bound to a membrane (receptor).
At the very least, the D-SLAM polypeptides can be used as molecular weight
markers
on SDS-PAGE gels or on molecular sieve gel filtration columns using methods
well known
to those of skill in the art. D-SLAM polypeptides can also be used to raise
antibodies, which
in turn are used to measure protein expression from a recombinant cell, as a
way of assessing
transformation of the host cell. Moreover, D-SLAM polypeptides can be used to
test the
following biological activities.
Gene Therapy Methods
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Another aspect of the present invention is to gene therapy methods for
treating,
diagnosing, detecting, and/or preventing disorders, diseases and/or
conditions. The gene
therapy methods relate to the introduction of nucleic acid (DNA, RNA and
antisense DNA or
RNA) sequences into an animal to achieve expression of the D-SLAM polypeptide
of the
present invention. This method requires a polynucleotide which codes for a D-
SLAM
polypeptide operatively linked to a promoter and any other genetic elements
necessary for the
expression of the polypeptide by the target tissue. Such gene therapy and
delivery techniques
are known in the art, see, for example, W090/11092, which is herein
incorporated by
reference.
Thus, for example, cells from a patient may be engineered with a
polynucleotide
(DNA or RNA) comprising a promoter operably linked to a D-SLAM polynucleotide
ex vivo,
with the engineered cells then being provided to a patient to be treated
(therapeutically and/or
prophylactically) and/or diagnosed with the polypeptide. Such methods are well-
known in
the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85:
207-216 (1993);
Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et
al., J.
Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-
229 (1995);
Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et
al., Human
Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255
(1997); and
Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 ( 1996)), which are herein
incorporated by
reference. In one embodiment, the cells which are engineered are arterial
cells. The arterial
cells may be reintroduced into the patient through direct injection to the
artery, the tissues
surrounding the artery, or through catheter injection.
As discussed in more detail below, the D-SLAM polynucleotide constructs can be
delivered by any method that delivers injectable materials to the cells of an
animal, such as,
injection into the interstitial space of tissues (heart, muscle, skin, lung,
liver, and the like).
The D-SLAM polynucleotide constructs may be delivered in a pharmaceutically
acceptable
liquid or aqueous carrier.
In one embodiment, the D-SLAM polynucleotide is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to
sequences that are
free from any delivery vehicle that acts to assist, promote or facilitate
entry into the cell,
including viral sequences, viral particles, liposome formulations, lipofectin
or precipitating
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agents and the like. However, the D-SLAM polynucleotides can also be delivered
in
liposome formulations and lipofectin formulations and the like can be prepared
by methods
well known to those skilled in the art. Such methods are described, for
example, in U.S.
Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated
by reference.
The D-SLAMpolynucleotide vector constructs used in the gene therapy method are
preferably constructs that will not integrate into the host genome nor will
they contain
sequences that allow for replication. Appropriate vectors include pWLNEO,
pSV2CAT,
pOG44, pXTI and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEFI/V5, pcDNA3.1, and pRc/CMV2 available from
Invitrogen. Other suitable vectors will be readily apparent to the skilled
artisan.
Any strong promoter known to those skilled in the art can be used for driving
the
expression of D-SLAM DNA. Suitable promoters include adenoviral promoters,
such as the
adenoviral major late promoter; or heterologous promoters, such as the
cytomegalovirus
(CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible
promoters, such
as the MMT promoter, the metallothionein promoter; heat shock promoters; the
albumin
promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase
promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-
actin
promoter; and human growth hormone promoters. The promoter also may be the
native
promoter for D-SLAM.
Unlike other gene therapy techniques, one major advantage of introducing naked
nucleic acid sequences into target cells is the transitory nature of the
polynucleotide synthesis
in the cells. Studies have shown that non-replicating DNA sequences can be
introduced into
cells to provide production of the desired polypeptide for periods of up to
six months.
The D-SLAM polynucleotide construct can be delivered to the interstitial space
of
tissues within the an animal, including of muscle, skin, brain, lung, liver,
spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,
and connective
tissue. Interstitial space of the tissues comprises the intercellular, fluid,
mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers in the
walls of vessels or
chambers, collagen fibers of fibrous tissues, or that same matrix within
connective tissue
ensheathing muscle cells or in the lacunae of bone. It is similarly the space
occupied by the
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plasma of the circulation and the lymph fluid of the lymphatic channels.
Delivery to the
interstitial space of muscle tissue is preferred for the reasons discussed
below. They may be
conveniently delivered by injection into the tissues comprising these cells.
They are
preferably delivered to and expressed in persistent, non-dividing cells which
are
differentiated, although delivery and expression may be achieved in non-
differentiated or less
completely differentiated cells, such as, for example, stem cells of blood or
skin fibroblasts.
In vivo muscle cells are particularly competent in their ability to take up
and express
polynucleotides.
For the naked acid sequence injection, an effective dosage amount of DNA or
RNA
will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg
body weight.
Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and
more preferably
from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary
skill will
appreciate, this dosage will vary according to the tissue site of injection.
The appropriate and
effective dosage of nucleic acid sequence can readily be determined by those
of ordinary skill
in the art and may depend on the condition being treated (therapeutically
and/or
prophylactically) and the route of administration.
The preferred route of administration is by the parenteral route of injection
into the
interstitial space of tissues. However, other parenteral routes may also be
used, such as,
inhalation of an aerosol formulation particularly for delivery to lungs or
bronchial tissues,
throat or mucous membranes of the nose. In addition, naked D-SLAM DNA
constructs can
be delivered to arteries during angioplasty by the catheter used in the
procedure.
The naked polynucleotides are delivered by any method known in the art,
including,
but not limited to, direct needle injection at the delivery site, intravenous
injection, topical
administration, catheter infusion, and so-called "gene guns". These delivery
methods are
known in the art.
As is evidenced in the Examples, naked D-SLAM nucleic acid sequences can be
administered in vivo results in the successful expression of D-SLAM
polypeptide in the
femoral arteries of rabbits.
The constructs may also be delivered with delivery vehicles such as viral
sequences,
viral particles, liposome formulations, lipofectin, precipitating agents, etc.
Such methods of
delivery are known in the art.
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In certain embodiments, the D-SLAM polynucleotide constructs are complexed in
a
liposome preparation. Liposomal preparations for use in the instant invention
include
cationic (positively charged), anionic (negatively charged) and neutral
preparations.
However, cationic liposomes are particularly preferred because a tight charge
complex can be
formed between the cationic liposome and the polyanionic nucleic acid.
Cationic liposomes
have been shown to mediate intracellular delivery of plasmid DNA (Felgner et
al., Proc. Natl.
Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by
reference); mRNA
(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is
herein
incorporated by reference); and purified transcription factors (Debs et al.,
J. Biol. Chem.
(1990) 265:10189-10192, which is herein incorporated by reference), in
functional form.
Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are
particularly
useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand
Island,
N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84:7413-
7416, which is
herein incorporated by reference). Other commercially available liposomes
include
transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
Other cationic liposomes can be prepared from readily available materials
using
techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092
(which is
herein incorporated by reference) for a description of the synthesis of DOTAP
(1,2-
bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA
liposomes
is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl.
Acad. Sci. USA
84:7413-7417, which is herein incorporated by reference. Similar methods can
be used to
prepare liposomes from other cationic lipid materials.
Similarly, anionic and neutral liposomes are readily available, such as from
Avanti
Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily
available materials.
Such materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine,
dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG),
dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can
also be mixed
with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for
making
liposomes using these materials are well known in the art.
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For example, commercially dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE) can
be used in various combinations to make conventional liposomes, with or
without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared
by drying
50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication
vial. The
sample is placed under a vacuum pump overnight and is hydrated the following
day with
deionized water. The sample is then sonicated for 2 hours in a capped vial,
using a Heat
Systems model 350 sonicator equipped with an inverted cup (bath type) probe at
the
maximum setting while the bath is circulated at 15EC. Alternatively,
negatively charged
vesicles can be prepared without sonication to produce multilamellar vesicles
or by extrusion
through nucleopore membranes to produce unilamellar vesicles of discrete size.
Other
methods are known and available to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar
vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being
preferred. The
various liposome-nucleic acid complexes are prepared using methods well known
in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527,
which is herein
incorporated by reference. For example, MLVs containing nucleic acid can be
prepared by
depositing a thin film of phospholipid on the walls of a glass tube and
subsequently hydrating
with a solution of the material to be encapsulated. SUVs are prepared by
extended sonication
of MLVs to produce a homogeneous population of unilamellar liposomes. The
material to be
entrapped is added to a suspension of preformed MLVs and then sonicated. When
using
liposomes containing cationic lipids, the dried lipid film is resuspended in
an appropriate
solution such as sterile water or an isotonic buffer solution such as 10 mM
Tris/NaCI,
sonicated, and then the preformed liposomes are mixed directly with the DNA.
The liposome
and DNA form a very stable complex due to binding of the positively charged
liposomes to
the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are
prepared by a
number of methods, well known in the art. Commonly used methods include CaZ+-
EDTA
chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al.,
Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A., Biochim.
Biophys. Acta
(1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836;
Fraley et al.,
Proc. Natl. Acad. Sci. USA (1979) 76:3348); detergent dialysis (Enoch, H. and
Strittmatter,
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126
P., Proc. Natl. Acad. Sci. USA ( 1979) 76:145); and reverse-phase evaporation
(REV) (Fraley
et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. and Papahadjopoulos, D.,
Proc. Natl. Acad.
Sci. USA (1978) 75:145; Schaefer-Ridder et al., Science (1982) 215:166), which
are herein
incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about
1:10.
Preferably, the ration will be from about S:1 to about 1:5. More preferably,
the ration will be
about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
U.S. Patent No. 5,676,954 (which is herein incorporated by reference) reports
on the
injection of genetic material, complexed with cationic liposomes carriers,
into mice. U.S.
Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859,
5,703,055, and international publication no. WO 94/9469 (which are herein
incorporated by
reference) provide cationic lipids for use in transfecting DNA into cells and
mammals. U.S.
Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international
publication no.
WO 94/9469 (which are herein incorporated by reference) provide methods for
delivering
DNA-cationic lipid complexes to mammals.
In certain embodiments, cells are be engineered, ex vivo or in vivo, using a
retroviral
particle containing RNA which comprises a sequence encoding D-SLAM.
Retroviruses from
which the retroviral plasmid vectors may be derived include, but are not
limited to, Moloney
Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey
Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form
producer cell lines. Examples of packaging cells which may be transfected
include, but are
not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE,
RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human
Gene
Therapy 1:5-14 ( 1990), which is incorporated herein by reference in its
entirety. The vector
may transduce the packaging cells through any means known in the art. Such
means include,
but are not limited to, electroporation, the use of liposomes, and CaP04
precipitation. In one
alternative, the retroviral plasmid vector may be encapsulated into a
liposome, or coupled to a
lipid, and then administered to a host.
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The producer cell line generates infectious retroviral vector particles which
include
polynucleotide encoding D-SLAM. Such retroviral vector particles then may be
employed,
to transduce eukaryotic cells, either in vitro or in vivo. The transduced
eukaryotic cells will
express D-SLAM.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with D-
SLAM
polynucleotide contained in an adenovirus vector. Adenovirus can be
manipulated such that
it encodes and expresses D-SLAM, and at the same time is inactivated in terms
of its ability
to replicate in a normal lytic viral life cycle. Adenovirus expression is
achieved without
integration of the viral DNA into the host cell chromosome, thereby
alleviating concerns
about insertional mutagenesis. Furthermore, adenoviruses have been used as
live enteric
vaccines for many years with an excellent safety profile (Schwartz, A. R. et
al. (1974) Am.
Rev. Respir. Dis.109:233-238). Finally, adenovirus mediated gene transfer has
been
demonstrated in a number of instances including transfer of alpha-1-
antitrypsin and CFTR to
the lungs of cotton rats (Rosenfeld, M., A. et al. (1991) Science 252:431-434;
Rosenfeld et al.,
(1992) Cell 68:143-155). Furthermore, extensive studies to attempt to
establish adenovirus as
a causative agent in human cancer were uniformly negative (Green, M. et al. (
1979) Proc.
Natl. Acad. Sci. USA 76:6606).
Suitable adenoviral vectors useful in the present invention are described, for
example,
in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 ( 1993); Rosenfeld
et al., Cell
68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al.,
Nature Genet. 7:362-369 ( 1994); Wilson et al., Nature 365:691-692 ( 1993);
and U.S. Patent
No. 5,652,224, which are herein incorporated by reference. For example, the
adenovirus
vector Ad2 is useful and can be grown in human 293 cells. These cells contain
the E1 region
of adenovirus and constitutively express Ela and Elb, which complement the
defective
adenoviruses by providing the products of the genes deleted from the vector.
In addition to
Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are also useful
in the present
invention.
Preferably, the adenoviruses used in the present invention are replication
deficient.
Replication deficient adenoviruses require the aid of a helper virus and/or
packaging cell line
to form infectious particles. The resulting virus is capable of infecting
cells and can express a
polynucleotide of interest which is operably linked to a promoter, for
example, the HARP
126
P., Proc. Natl. Acad. Sci. US
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promoter of the present invention, but cannot replicate in most cells.
Replication deficient
adenoviruses may be deleted in one or more of all or a portion of the
following genes: Ela,
Elb, E3, E4, E2a, or L1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo,
using an
adeno-associated virus (AAV). AAVs are naturally occurring defective viruses
that require
helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in
Microbiol.
Immunol. 158:97 ( 1992)). It is also one of the few viruses that may integrate
its DNA into
non-dividing cells. Vectors containing as little as 300 base pairs of AAV can
be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods
for
producing and using such AAVs are known in the art. See, for example, U.S.
Patent Nos.
5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and
5,589,377.
For example, an appropriate AAV vector for use in the present invention will
include
all the sequences necessary for DNA replication, encapsidation, and host-cell
integration.
The D-SLAM polynucleotide construct is inserted into the AAV vector using
standard
cloning methods, such as those found in Sambrook et al., Molecular Cloning: A
Laboratory
Manual, Cold Spring Harbor Press ( 1989). The recombinant AAV vector is then
transfected
into packaging cells which are infected with a helper virus, using any
standard technique,
including lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate
helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or
herpes viruses.
Once the packaging cells are transfected and infected, they will produce
infectious AAV viral
particles which contain the D-SLAM polynucleotide construct. These viral
particles are then
used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced
cells will contain
the D-SLAM polynucleotide construct integrated into its genome, and will
express D-SLAM.
Another method of gene therapy involves operably associating heterologous
control
regions and endogenous polynucleotide sequences (e.g. encoding D-SLAM) via
homologous
recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997;
International
Publication No. WO 96/29411, published September 26, 1996; International
Publication No.
WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci.
USA
86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989). This
method involves
the activation of a gene which is present in the target cells, but which is
not normally
expressed in the cells, or is expressed at a lower level than desired.
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Polynucleotide constructs are made, using standard techniques known in the
art,
which contain the promoter with targeting sequences flanking the promoter.
Suitable
promoters are described herein. The targeting sequence is sufficiently
complementary to an
endogenous sequence to permit homologous recombination of the promoter-
targeting
sequence with the endogenous sequence. The targeting sequence will be
sufficiently near the
5' end of the D-SLAM desired endogenous polynucleotide sequence so the
promoter will be
operably linked to the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR.
Preferably,
the amplified promoter contains distinct restriction enzyme sites on the 5'
and 3' ends.
Preferably, the 3' end of the first targeting sequence contains the same
restriction enzyme site
as the 5' end of the amplified promoter and the 5' end of the second targeting
sequence
contains the same restriction site as the 3' end of the amplified promoter.
The amplified
promoter and targeting sequences are digested and ligated together.
The promoter-targeting sequence construct is delivered to the cells, either as
naked
polynucleotide, or in conjunction with transfection-facilitating agents, such
as liposomes,
viral sequences, viral particles, whole viruses, lipofection, precipitating
agents, etc., described
in more detail above. The P promoter-targeting sequence can be delivered by
any method,
included direct needle injection, intravenous injection, topical
administration, catheter
infusion, particle accelerators, etc. The methods are described in more detail
below.
The promoter-targeting sequence construct is taken up by cells. Homologous
recombination between the construct and the endogenous sequence takes place,
such that an
endogenous D-SLAM sequence is placed under the control of the promoter. The
promoter
then drives the expression of the endogenous D-SLAM sequence.
The polynucleotides encoding D-SLAM may be administered along with other
polynucleotides encoding other angiongenic proteins. Angiogenic proteins
include, but are
not limited to, acidic and basic fibroblast growth factors, VEGF-1, epidermal
growth factor
alpha and beta, platelet-derived endothelial cell growth factor, platelet-
derived growth factor,
tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth
factor, colony
stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony
stimulating factor, and nitric oxide synthase.
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Preferably, the polynucleotide encoding D-SLAM contains a secretory signal
sequence that facilitates secretion of the protein. Typically, the signal
sequence is positioned
in the coding region of the polynucleotide to be expressed towards or at the
5' end of the
coding region. The signal sequence may be homologous or heterologous to the
polynucleotide of interest and may be homologous or heterologous to the cells
to be
transfected. Additionally, the signal sequence may be chemically synthesized
using methods
known in the art.
Any mode of administration of any of the above-described polynucleotides
constructs
can be used so long as the mode results in the expression of one or more
molecules in an
amount sufficient to provide a therapeutic effect. This includes direct needle
injection,
systemic injection, catheter infusion, biolistic injectors, particle
accelerators (i.e., "gene
guns"), gelfoam sponge depots, other commercially available depot materials,
osmotic pumps
(e.g., Alza minipumps), oral or suppositorial solid (tablet or pill)
pharmaceutical
formulations, and decanting or topical applications during surgery. For
example, direct
injection of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a
protein-coated plasmid into the portal vein has resulted in gene expression of
the foreign gene
in the rat livers (Kaneda et al., Science 243:375 (1989)).
A preferred method of local administration is by direct injection. Preferably,
a
recombinant molecule of the present invention complexed with a delivery
vehicle is
administered by direct injection into or locally'within the area of arteries.
Administration of a
composition locally within the area of arteries refers to injecting the
composition centimeters
and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide
construct of the
present invention in or around a surgical wound. For example, a patient can
undergo surgery
and the polynucleotide construct can be coated on the surface of tissue inside
the wound or
the construct can be injected into areas of tissue inside the wound.
Therapeutic compositions useful in systemic administration, include
recombinant
molecules of the present invention complexed to a targeted delivery vehicle of
the present
invention. Suitable delivery vehicles for use with systemic administration
comprise
liposomes comprising ligands for targeting the vehicle to a particular site.
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Preferred methods of systemic administration, include intravenous injection,
aerosol,
oral and percutaneous (topical) delivery. Intravenous injections can be
performed using
methods standard in the art. Aerosol delivery can also be performed using
methods standard
in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA
189:11277-11281,
1992, which is incorporated herein by reference). Oral delivery can be
performed by
complexing a polynucleotide construct of the present invention to a carrier
capable of
withstanding degradation by digestive enzymes in the gut of an animal.
Examples of such
carriers, include plastic capsules or tablets, such as those known in the art.
Topical delivery
can be performed by mixing a polynucleotide construct of the present invention
with a
lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend upon a
number of factors including, for example, the chemical structure and
biological activity of the
substance, the age and weight of the animal, the precise condition requiring
treatment and its
severity, and the route of administration. The frequency of treatments depends
upon a
number of factors, such as the amount of polynucleotide constructs
administered per dose, as
well as the health and history of the subject. The precise amount, number of
doses, and
timing of doses will be determined by the attending physician or veterinarian.
Therapeutic compositions of the present invention can be administered to any
animal,
preferably to mammals and birds. Preferred mammals include humans, dogs, cats,
mice, rats,
rabbits sheep, cattle, horses and pigs, with humans being particularly
preferred.
Biological Activities of D-SLAM
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
can
be used in assays to test for one or more biological activities. If D-SLAM
polynucleotides or
polypeptides, or agonists or antagonists of D-SLAM, do exhibit activity in a
particular assay,
it is likely that D-SLAM may be involved in the diseases associated with the
biological
activity. Therefore, D-SLAM could be used to treat, diagnose, detect, and/or
prevent the
associated disease.
D-SLAM is a cell surface receptor homologous to members of the Secreted
Lymphocyte Activation Molecule (SLAM) family, and thus should have activity
similar to
other SLAM family members. Current studies in the literature demonstrate that
SLAM can
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associate with itself, and that this homotypic interaction can activate B- and
T-cells.
Therefore, D-SLAM may interact specifically with SLAM, with D-SLAM (a
homotypic
interaction), or other B- and T-cell receptor molecules on the surface of B-
and T-cells to
affect the activation, proliferation, survival, and/or differentiation of
immune cells. Similarly,
soluble D-SLAM may be an important costimulatory molecule for therapeutic uses
or
immune modulation. Ligands, such as antibodies, may mimic the action of
soluble D-SLAM
by binding to D-SLAM, SLAM, or other dendritic cell receptors.
Binding of D-SLAM induces the production of interferon-gamma from other cell
types, particularly T- and B-cells (data not shown.) The binding may occur
through
homotypic association with membrane bound D-SLAM, association with SLAM, or
association with other T- or B-cell receptors. Ligands, such as antibodies,
may mimic the
induction of interferon-gamma by soluble D-SLAM by binding to D-SLAM, SLAM, or
other
dendritic cell receptors.
Moreover, because of the tissue distribution of D-SLAM, this protein may also
play a
role in stimulating dendritic or antigen presenting cells. For example, a
secreted form of D-
SLAM, containing the extracellular domain or the full-length form, may bind to
and stimulate
D-SLAM molecules located on the surface of dendritic or antigen-presenting
cells in
homotypic manner. Binding may also occur to SLAM, or other dendritic cell
surface
receptors. This binding may regulate the survival, proliferation,
differentiation, activation or
maturation of dendritic cells or antigen presenting cells, effecting antigen
recognition and
immune response. Moreover, ligands, such as antibodies, may mimic the action
of soluble D-
SLAM by binding to D-SLAM, SLAM, or other dendritic cell receptors.
Thus, D-SLAM may be useful as a therapeutic molecule. It could be used to
control
the proliferation, activation, maturation, survival, and/or differentiation of
hematopoietic
cells, in particular B- and T-cells. Particularly, D-SLAM may be a useful
therapeutic to
mediate immune modulation, and may influence the Th0-TH1-TH2 profile of a
patient's
immune system. For example, D-SLAM may drive immune response to the Th0-TH 1
pathway. This control of immune cells would be particularly important in the
treatment,
diagnosis, detection, and/or prevention of immune disorders, such as
autoimmune diseases or
immunosuppression (see below). Preferably, treatment, diagnosis, detection,
and/or
prevention of immune disorders could be carried out using a secreted form of D-
SLAM, gene
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therapy, or ex vivo applications. Moreover, inhibitors of D-SLAM , either
blocking
antibodies or mutant forms, could modulate the expression of D-SLAM. These
inhibitors
may be useful to treat, diagnose, detect, and/or prevent diseases associated
with the
misregulation of D-SLAM, such as T cell lymphoma.
In one embodiment, the invention provides a method for the specific delivery
of
compositions of the invention to cells by administering polypeptides of the
invention (e.g., D-
SLAM polypeptides or anti-D-SLAM antibodies) 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.
In another embodiment, the invention provides a method for the specific
destruction
of cells (e.g., the destruction of tumor cells) by administering polypeptides
of the invention
(e.g., D-SLAM polypeptides or anti-D-SLAM antibodies) in association with
toxins or
cytotoxic prodrugs.
By "toxin" is meant compounds that bind and activate endogenous cytotoxic
effector
systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of
toxins, cytotoxins
(cytotoxic agents), or any molecules or enzymes not normally present in or on
the surface of a
cell that under defined conditions cause the cell's death. Toxins that may be
used according
to the methods of the invention include, but are not limited to, radioisotopes
known in the art,
compounds such as, for example, antibodies (or complement fixing containing
portions
thereof) that bind an inherent or induced endogenous cytotoxic effector
system, thymidine
kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin
A, diphtheria
toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin
and cholera
toxin. "Toxin" also includes a cytostatic or cytocidal agent, a therapeutic
agent or a
radioactive metal ion, e.g., alpha-emitters such as, for example, '''3Bi, or
other radioisotopes
such as, for example, '°3Pd, '33Xe, "'I, 6~Ge, 5'Co, ~SZn, 85Sr, 3ZP,
355, y"Y, 'S'Sm, 'S3Gd, '~yYb, 5'Cr,
S~Mn, 'SSe, "3Sn, y"Yttrium, "'Tin, 'g~Rhenium, '~~Holmium, and '~xRhenium;
luminescent
labels, such as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
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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). A cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells. Examples
include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone,
glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin
and analogs or
homologs thereof. 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), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)),
and anti-mitotic agents (e.g., vincristine and vinblastine).
By "cytotoxic prodrug" is meant a non-toxic compound that is converted by an
enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic
prodrugs that
may be used according to the methods of the invention include, but are not
limited to,
glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate
derivatives of
etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and
phenoxyacetamide
derivatives of doxorubicin.
It will be appreciated that conditions caused by a decrease in the standard or
normal
level of D-SLAM activity in an individual, particularly disorders of the
immune system, can
be treated by administration of D-SLAM polypeptide (in the form of soluble
extracellular
domain or cells expressing the complete protein) or agonist. Thus, the
invention also
provides a method of treatment of an individual in need of an increased level
of D-SLAM
activity comprising administering to such an individual a pharmaceutical
composition
comprising an amount of an isolated D-SLAM polypeptide of the invention, or
agonist
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thereof (e.g, an agonistic D-SLAM antibody), effective to increase the D-SLAM
activity level
in such an individual.
It will also be appreciated that conditions caused by a increase in the
standard or
normal level of D-SLAM activity in an individual, particularly disorders of
the immune
system, can be treated by administration of D-SLAM polypeptides (in the form
of soluble
extracellular domain or cells expressing the complete protein) or antagonist
(e.g, an
antagonistic D-SLAM antibody). Thus, the invention also provides a method of
treatment of
an individual in need of an dereased level of D-SLAM activity comprising
administering to
such an individual a pharmaceutical composition comprising an amount of an
isolated D-
SLAM polypeptide of the invention, or antagonist thereof, effective to
decrease the D-SLAM
activity level in such an individual.
Viruses are one example of an infectious agent that can cause disease or
symptoms
that can be treated by D-SLAM polynucleotides or polypeptides, or agonists of
D-SLAM.
Examples of viruses, include, but are not limited to the following DNA and RNA
viruses and
viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,
Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV,
Flaviviridae,
Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes
Simplex,
Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae),
Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma
virus,
Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or
Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and
Togaviridae
(e.g., Rubivirus). Viruses falling within these families can cause a variety
of diseases or
symptoms, including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial virus,
encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic
fatigue syndrome, hepatitis
(A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift
Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS),
pneumonia,
Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza,
Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted
diseases, skin
diseases (e.g., Kaposi's, warts), and viremia. D-SLAM polynucleotides or
polypeptides, or
agonists or antagonists of D-SLAM, can be used to treat, prevent, diagnose,
and/or detect any
of these symptoms or diseases. In specific embodiments, D-SLAM
polynucleotides,
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polypeptides, or agonists are used to treat, prevent, and/or diagnose:
meningitis, Dengue,
EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific
embodiment D-SLAM
polynucleotides, polypeptides, or agonists are used to treat patients
nonresponsive to one or
more other commercially available hepatitis vaccines. In a further specific
embodiment, D-
SLAM polynucleotides, polypeptides, or agonists are used to treat, prevent,
and/or diagnose
AIDS. In an additional specific embodiment D-SLAM polynucleotides,
polypeptides,
agonists, and/or antagonists are used to treat, prevent, and/or diagnose
patients with
cryptosporidiosis.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that can
be treated by D-SLAM polynucleotides or polypeptides, or agonists or
antagonists of D
SLAM, include, but not limited to, the following Gram-Negative and Gram-
positive bacteria
and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium,
Mycobacterium,
Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,
Anthrax,
Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g.,
Borrelia burgdorferi,
Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,
Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic
E. coli),
Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and
Salmonella
paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis,
Leptospirosis,
Listeria (e.g, Listeria monocytogenes), Mycoplasmatales, Mycobacterium leprae,
Vibrio
cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal),
Meisseria
meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus
(e.g., Heamophilus
influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae,
Syphilis,
Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal
(e.g.,
Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal
families
can cause the following diseases or symptoms, including, but not limited to:
bacteremia,
endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), .
gingivitis, opportunistic
infections (e.g., AIDS related infections), paronychia, prosthesis-related
infections, Reiter's
Disease, respiratory tract infections, such as Whooping Cough or Empyema,
sepsis, Lyme
Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,
Typhoid,
pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia,
Syphilis,
Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism,
gangrene, tetanus,
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impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g.,
cellulitis, dermatocycoses), toxemia, urinary tract infections, wound
infections. D-SLAM
polynucleotides or polypeptides, or agonists or antagonists of D-SLAM, can be
used to treat,
prevent, diagnose, and/or detect any of these symptoms or diseases. In
specific embodiments,
S D-SLAM polynucleotides, polypeptides, or antagonists thereof are used to
treat, prevent,
and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated by
D-
SLAM polynucleotides or polypeptides, or antagonists of D-SLAM, include, but
not limited
to, the following families or class: Amebiasis, Babesiosis, Coccidiosis,
Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis,
Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans
(e.g.,
Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium
ovate).
These parasites can cause a variety of diseases or symptoms, including, but
not limited to:
Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,
giardiasis), liver
disease, lung disease, opportunistic infections (e.g., AIDS related), malaria,
pregnancy
complications, and toxoplasmosis. D-SLAM polynucleotides or polypeptides, or
antagonists
or antagonists of D-SLAM, can be used to treat, prevent, diagnose, and/or
detect any of these
symptoms or diseases. In specific embodiments, D-SLAM polynucleotides,
polypeptides, or
antagonists thereof are used to treat, prevent, and/or diagnose malaria.
In another embodiment, D-SLAM polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or
diagnose inner
ear infection (such as, for example, otitis media), as well as other
infections characterized by
infection with Streptococcus pneumoniae and other pathogenic organisms.
In a specific embodiment, D-SLAM polynucleotides or polypeptides, or agonists
or
antagonists thereof (e.g., anti-D-SLAM antibodies) are used to treat or
prevent a disorder
characterized by deficient serum immunoglobulin production, recurrent
infections, and/or
immune system dysfunction. Moreover, D-SLAM polynucleotides or polypeptides,
or
agonists or antagonists thereof (e.g., anti-D-SLAM antibodies) 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
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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
pheumocystis carnii.
D-SLAM polynucleotides or polypeptides of the invention, or agonists or
antagonists
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.
Additionally, D-SLAM polynucleotides or polypeptides of the invention, or
agonists
or antagonists thereof, may be used to diagnose, prognose, 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
mutliple 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,
cytamegalovirus (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.
Additional preferred embodiments of the invention include, but are not limited
to, the
use of D-SLAM polypeptides, D-SLAM polynucleotides, and functional agonists
thereof, in
the following applications:
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 inhibit the immune system to produce decreased quantities
of one or
more antibodies (e.g., IgG, IgA, IgM, and IgE), to inhibit higher affinity
antibody production
(e.g., IgG, IgA, IgM, and IgE), and/or to decrease an immune response. In a
specific
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nonexclusive embodiment, D-SLAM polypeptides of the invention, and/or agonists
thereof,
are administered to inhibit the immune system to produce decreased quantities
of IgG. In
another specific nonexclusive embodiment, D-SLAM polypeptides of the invention
and/or
agonists thereof, are administered to boost the immune system to produce
decreased
quantities of IgA. In another specific nonexclusive embodiment, D-SLAM
polypeptides of
the invention and/or agonists thereof, are administered to inhibit the immune
system to
produce decreased quantities of IgM.
As an agent that reduces the immune status of an individual prior to their
receipt of
immunosuppressive therapies.
As an agent to decrease serum immunoglobulin concentrations.
As an immune system inhibitor 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.
As an agent to reduce immunoresponsiveness. B cell immunodeficiencies that may
be ameliorated or treated by administering D-SLAM antagonists 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
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
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(XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte
Syndrome)
and severe combined immunodeficiency.
D-SLAM antagonists may be used as agents 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
the D-SLAM
antagonists of the invention include, but are not limited to, HIV Infection,
AIDS, bone
marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
D-SLAM antagonists may be used as agents 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 D-SLAM
antagonists
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.
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.
As a means to inhibit tumor proliferation.
As a therapy for generation and/or regeneration of lymphoid tissues following
surgery, trauma or genetic defect.
As a gene-based therapy for genetically inherited disorders resulting in
immuno-
incompetence such as observed among SLID patients.
As an antigen for the generation of antibodies to inhibit or enhance D-SLAM-
mediated responses.
As a means of inhibiting monocytes/macrophages to defend against parasitic
diseases
that effect monocytes such as Leshmania.
As pretreatment of bone marrow samples prior to transplant. Such treatment
would
decrease B cell representation and thus modulate recovery.
As a means of regulating secreted cytokines that are elicited by D-SLAM.
D-SLAM polypeptides or polynucleotides of the invention, or antagonists may be
used to modulate IgE concentrations in vitro or in vivo.
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Additionally, D-SLAM polypeptides or polynucleotides of the invention, or
antagonists thereof, 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.
In a specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention,
or antagonists thereof, is administered to treat, prevent, diagnose, and/or
ameliorate selective
IgA deficiency.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent, .
diagnose, and/or
ameliorate ataxia-telangiectasia.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent,
diagnose, and/or
ameliorate common variable immunodeficiency.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent,
diagnose, and/or
ameliorate X-linked agammaglobulinemia.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent,
diagnose, and/or
ameliorate severe combined immunodeficiency (SCID).
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent,
diagnose, and/or
ameliorate Wiskott-Aldrich syndrome.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent,
diagnose, and/or
ameliorate X-linked Ig deficiency with hyper IgM.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists or antagonists (e.g., anti-D-SLAM antibodies)
thereof, is
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.
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In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent,
diagnose, and/or
ameliorate monocytic leukemoid reaction, as seen, for example, with
tuberculosis.
In another specific embodiment, D-SLAM polypeptides or polynucleotides of the
invention, or antagonists thereof, is administered to treat, prevent,
diagnose, and/or
ameliorate monocytic leukocytosis, monocytic leukopenia, monocytopenia, and/or
monocytosis.
In a specific embodiment, D-SLAM polynucleotides or polypeptides of the
invention,
and/or anti-D-SLAM antibodies and/or agonists or antagonists thereof, are used
to treat,
prevent, detect, and/or diagnose primary B lymphocyte disorders and/or
diseases, and/or
conditions associated therewith.
In a preferred embodiment, D-SLAM polynucleotides, polypeptides, and/or
agonists
and/or antagonists thereof 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
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
mucopolysaccharidosis), 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),
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mucous colitis (such as, for example, mucocolitis and myxomembi-anous
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, D-SLAM polynucleotides, polypeptides, and/or
agonists
and/or antagonists thereof are used to treat, prevent, and/or diagnose
mucositis, especially as
associated with chemotherapy.
In a preferred embodiment, D-SLAM polynucleotides, polypeptides, and/or
agonists
and/or antagonists thereof are used to treat, prevent, and/or diagnose
diseases or disorders
affecting or conditions associated with sinusitis.
An additional condition, disease or symptom that can be treated, prevented,
and/or
diagnosed by D-SLAM polynucleotides or polypeptides, or antagonists of D-SLAM,
is
osteomyelitis.
An additional condition, disease or symptom that can be treated, prevented,
and/or
diagnosed by D-SLAM polynucleotides or polypeptides, or antagonists of D-SLAM,
is
endocarditis.
All of the above described applications as they may apply to veterinary
medicine.
D-SLAM antagonists may be used as a therapy for B cell malignancies such as
ALL,
Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia,
plasmacytomas,
multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases, as well as
a therapy
for chronic hypergammaglobulinemeia evident in such diseases as
monoclonalgammopathy
of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic
monoclonalgammopathies, and plasmacytomas.
An immunosuppressive agent(s).
D-SLAM polypeptides or polynucleotides of the invention, or antagonists may be
used to modulate IgE concentrations in vitro or in vivo.
In another embodiment, administration of D-SLAM polypeptides or
polynucleotides
of the invention, or antagonists thereof, may be used to treat, prevent,
and/or diagnose IgE-
mediated allergic reactions including, but not limited to, asthma, rhinitis,
and eczema.
The above-recited applications have uses in a wide variety of hosts. Such
hosts
include, but are not limited to, human, murine, rabbit, goat, guinea pig,
camel, horse, mouse,
rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human
primate, and
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human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig,
chicken, rat,
hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a
mammal. In most
preferred embodiments, the host is a human.
The agonists and antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as described herein.
All of the above described applications as they may apply to veterinary
medicine.
Moreover, all applications described herein may also apply to veterinary
medicine.
D-SLAM polynucleotides or polypeptides of the invention and/or agonists and/or
antagonists thereof, may be used to treat, prevent, and/or diagnose various
immune
system-related disorders and/or conditions associated with these disorders, in
mammals,
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 D-
SLAM polynucleotides or polypeptides of the invention and/or agonists and/or
agonists
thereof 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, D-SLAM agonists of the
invention
are used to treat, prevent, and/or diagnose an autoimmune disorder.
Autoimmune disorders and conditions associated with these disorders that may
be
treated, prevented, and/or diagnosed with the D-SLAM polynucleotides,
polypeptides, and/or
agonists of the invention, include, but are not limited to, autoimmune
hemolytic anemia,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis,
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, Guillain-Barre Syndrome,
insulin
dependent diabetes mellitis, and autoimmune inflammatory eye disease.
Additional autoimmune disorders (that are highly probable) that may be
treated,
prevented, and/or diagnosed with the compositions of the invention include,
but are not
limited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis) (often
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characterized, e.g., by cell-mediated and humoral thyroid cytotoxicity),
systemic lupus
erhythematosus (often characterized, e.g., by circulating and locally
generated immune
complexes), 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 (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.
Additional autoimmune disorders (that are probable) that may be treated,
prevented,
and/or diagnosed with the 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, andlor 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).
Additional autoimmune disorders (that are possible) that may be treated,
prevented,
and/or diagnosed with the compositions of the invention include, but are not
limited to,
chronic active hepatitis (often characterized, e.g., by smooth muscle
antibodies), primary
biliary cirrhosis (often characterized, e.g., by mitchondrial antibodies),
other endocrine gland
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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), inflammatory
myopathies, and
many other inflammatory, granulamatous, degenerative, and atrophic disorders.
In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions
associated with the diseases and disorders recited above are treated,
prevented, and/or
diagnosed using anti-D-SLAM antibodies.
In a specific preferred embodiment, rheumatoid arthritis is treated,
prevented, and/or
diagnosed using D-SLAM and/or other agonists of the invention.
In a specific preferred embodiment, lupus is treated, prevented, and/or
diagnosed
using D-SLAM and/or other agonists of the invention.
In a specific preferred embodiment, nephritis associated with lupus is
treated,
prevented, and/or diagnosed using D-SLAM and/or other agonists of the
invention.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic
asthma) or other respiratory problems, may also be treated by D-SLAM
polynucleotides or
polypeptides of the invention and/or agonists and/or antagonists thereof.
Moreover, these
molecules can be used to treat, prevent, and/or diagnose anaphylaxis,
hypersensitivity to an
antigenic molecule, or blood group incompatibility.
D-SLAM polynucleotides or polypeptides of the invention and/or agonists and/or
antagonists thereof, may also be used to treat, prevent, and/or diagnose organ
rejection or
graft-versus-host disease (GVHD) 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 D-SLAM
polynucleotides or polypeptides of the invention and/or agonists and/or
antagonists thereof,
that inhibits an immune response, particularly the proliferation,
differentiation, or chemotaxis
of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
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Similarly, D-SLAM polynucleotides or polypeptides of the invention and/or
agonists
and/or antagonists thereof, may also be used to modulate inflammation. For
example, D-
SLAM polynucleotides or polypeptides of the invention and/or agonists and/or
antagonists
thereof, may inhibit the proliferation and differentiation of cells involved
in an inflammatory
response. These molecules can be used to treat, prevent, and/or diagnose
inflammatory
conditions, both chronic and acute conditions, including chronic prostatitis,
granulomatous
prostatitis and malacoplakia, inflammation associated with infection (e.g.,
septic shock,
sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-
reperfusion injury,
endotoxin lethality, arthritis, complement-mediated hyperacute rejection,
nephritis, cytokine
or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease,
or resulting
from over production of cytokines (e.g., TNF or IL-1.)
In a specific embodiment, anti-D-SLAM antibodies of the invention are used to
treat,
prevent, modulate, detect, and/or diagnose inflammation.
In a specific embodiment, anti-D-SLAM antibodies of the invention are used to
treat,
prevent, modulate, detect, and/or diagnose inflamatory disorders.
In another specific embodiment, anti-D-SLAM antibodies of the invention are
used to
treat, prevent, modulate, detect, and/or diagnose allergy and/or
hypersensitivity.
In a specific embodiment, D-SLAM polynucleotides or polypeptides of the
invention
and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or
diagnose chronic
obstructive pulmonary disease (COPD).
In another embodiment, D-SLAM polynucleotides or polypeptides of the invention
and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or
diagnose fibroses
and conditions associated with fibroses, such as, for example, 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 retroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal
fibrosis, nodular
subepidermal fibrosis, pericentral fibrosis, perimuscular fibrosis, pipestem
fibrosis,
replacement fibrosis, subadventitial fibrosis, and Symmers' clay pipestem
fibrosis.
Diseases associated with increased cell survival, or the inhibition of
apoptosis that
may be diagnosed, treated, or prevented with the D-SLAM polynucleotides or
polypeptides
of the invention, and agonists and antagonists thereof, include cancers (such
as follicular
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lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,
including, but
not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma,
glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach
cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma,
osteoclastoma,
osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer,
Kaposi's sarcoma
and ovarian cancer); autoimmune disorders (such as systemic lupus
erythematosus and
immune-related glomerulonephritis rheumatoid arthritis); viral infections
(such as herpes
viruses, pox viruses and adenoviruses); inflammation; graft vs. host disease;
acute graft
rejection and chronic graft rejection. Thus, in preferred embodiments D-SLAM
polynucleotides or polypeptides of the invention abd/or agonists or
antagonists thereof, are
used to treat, prevent, and/or diagnose autoimmune diseases and/or inhibit the
growth,
progression, and/or metastasis of cancers, including, but not limited to,
those cancers
disclosed herein, such as, for example, lymphocytic leukemias (including, for
example, MLL
and chronic lymphocytic leukemia (CLL)) and follicular lymphomas. In another
embodiment
D-SLAM polynucleotides or polypeptides of the invention are used to activate,
differentiate
or proliferate cancerous cells or tissue (e.g., B cell lineage related cancers
(e.g., CLL and
MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more
vulnerable to
cancer therapy (e.g., chemotherapy or radiation therapy).
Moreover, in other embodiments, D-SLAM polynucleotides or polypeptides of the
invention or agonists or antagonists thereof, are used to inhibit the growth,
progression,
and/or metastases of malignancies and related disorders such as leukemia
(including acute
leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia
(including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia))
and
chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic
leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-
Hodgkin's
disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, and
solid tumors including, but not limited to, sarcomas and carcinomas such as
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,
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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, Wilm's 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, menangioma, melanoma,
neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis apoptosis that may be diagnosed,
treated, or prevented with the D-SLAM polynucleotides or polypeptides of the
invention, and
agonists and antagonists thereof, include AIDS; neurodegenerative disorders
(such as
Alzheimer's disease, Parkinson's disease, 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.
Thus, in preferred embodiments D-SLAM polynucleotides or polypeptides of the
invention
and/or agonists or antagonists thereof, are used to treat, prevent, and/or
diagnose the diseases
and disorders listed above.
Polynucleotides and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are useful in the diagnosis and treatment or prevention of
a wide range of
diseases and/or conditions. Such diseases and conditions include, but are not
limited to,
cancer (e.g., immune cell related cancers, breast cancer, prostate cancer,
ovarian cancer,
follicular lymphoma, cancer associated with mutation or alteration of p53,
brain tumor,
bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-
small cell
carcinoma of the lung, small cell carcinoma of the lung, stomach cancer,
etc.),
lymphoproliferative disorders (e.g., lymphadenopathy), microbial (e.g., viral,
bacterial, etc.)
infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirus infection
(including, but not
limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection,
poxvirus infection, human papilloma virus infection, hepatitis infection
(e.g., HAV, HBV,
HCV, etc.), Helicobacter pylori infection, invasive Staphylococcia, etc.),
parasitic infection,
nephritis, bone disease (e.g., osteoporosis), atherosclerosis, pain,
cardiovascular disorders
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(e.g., neovascularization, hypovascularization or reduced circulation (e.g.,
ischemic disease
(e.g., myocardial infarction, stroke, etc.)), AIDS, allergy, inflammation,
neurodegenerative
disease (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis,
pigmentary retinitis, cerebellar degeneration, etc.), graft rejection (acute
and chronic), graft
vs. host disease, diseases due to osteomyelodysplasia (e.g., aplastic anemia,
etc.), joint tissue
destruction in rheumatism, liver disease (e.g., acute and chronic hepatitis,
liver injury, and
cirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoid
arthritis, systemic lupus
erythematosus, immune complex glomerulonephritis, autoimmune diabetes,
autoimmune
thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis, etc.),
cardiomyopathy
(e.g., dilated cardiomyopathy), diabetes, diabetic complications (e.g.,
diabetic nephropathy,
diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis,
glomerulonephritis,
septic shock, and ulcerative colitis.
Polynucleotides and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are useful in promoting angiogenesis, wound healing (e.g.,
wounds,
burns, and bone fractures). Polynucleotides and/or polypeptides of the
invention and/or
agonists and/or antagonists thereof are also useful as an adjuvant to enhance
immune
responsiveness to specific antigen, anti-viral immune responses.
More generally, polynucleotides and/or polypeptides of the invention and/or
agonists
and/or antagonists thereof are useful in regulating (i.e., elevating or
reducing) immune
response. For example, polynucleotides and/or polypeptides of the invention
may be useful
in preparation or recovery from surgery, trauma, radiation therapy,
chemotherapy, and
transplantation, or may be used to boost immune response and/or recovery in
the elderly and
immunocompromised individuals. Alternatively, polynucleotides and/or
polypeptides of the
invention and/or agonists and/or antagonists thereof are useful as
immunosuppressive agents,
for example in the treatment or prevention of autoimmune disorders. In
specific
embodiments, polynucleotides and/or polypeptides of the invention are used to
treat or
prevent chronic inflammatory, allergic or autoimmune conditions, such as those
described
herein or are otherwise known in the art.
Preferably, treatment using D-SLAM polynucleotides or polypeptides, and/or
agonists
or antagonists of D-SLAM (e.g., anti-D-SLAM antibody), could either be by
administering
an effective amount of D-SLAM polypeptide of the invention, or agonist or
antagonist
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thereof, to the patient, or by removing cells from the patient, supplying the
cells with D-
SLAM polynucleotide, and returning the engineered cells to the patient (ex
vivo therapy).
Moreover, as further discussed herein, the D-SLAM polypeptide or
polynucleotide can be
used as an adjuvant in a vaccine to raise an immune response against
infectious disease.
Immune Activity
In one embodiment, D-SLAM polynucleotides or polypeptides or D-SLAM agonists
or antagonists (e.g., anti-D-SLAM antibodies) of the invention are used to
treat, prevent,
diagnose, or prognose an individual having an immunodeficiency.
Immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed
with
the D-SLAM polynucleotides or polypeptides or D-SLAM agonists or antagonists
(e.g., anti-
D-SLAM antibodies) of the invention, include, but are not limited to one or
more
immunodeficiencies selected from: 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.
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D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may be useful in treating, diagnosing, detecting, and/or preventing
deficiencies or disorders
of the immune system, by activating or inhibiting the proliferation,
differentiation, or
mobilization (chemotaxis) of immune cells. Immune cells develop through a
process called
hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and
macrophages)
and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The
etiology of these
immune deficiencies or disorders may be genetic, somatic, such as cancer or
some
autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or
infectious. Moreover,
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
can be
used as a marker or detector of a particular immune system disease or
disorder.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may be useful in treating, diagnosing, detecting, and/or preventing
deficiencies or disorders
of hematopoietic cells. D-SLAM polynucleotides or polypeptides, or agonists or
antagonists
of D-SLAM, could be used to increase differentiation and proliferation of
hematopoietic
cells, including the pluripotent stem cells, in an effort to treat, diagnose,
detect, and/or
prevent those disorders associated with a decrease in certain (or many) types
hematopoietic
cells. Examples of immunologic deficiency syndromes include, but are not
limited to: blood
protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia
telangiectasia,
common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV
infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte
bactericidal
dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich
Disorder,
anemia, thrombocytopenia, or hemoglobinuria.
Moreover, D-SLAM polynucleotides or polypeptides, or agonists or antagonists
of D
SLAM, can also be used to modulate hemostatic (the stopping of bleeding) or
thrombolytic
activity (clot formation). For example, by increasing hemostatic or
thrombolytic activity, D
SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
could be
used to treat, diagnose, detect, and/or prevent blood coagulation disorders
(e.g.,
afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. -
thrombocytopenia), or
wounds resulting from trauma, surgery, or other causes. Alternatively, D-SLAM
polynucleotides or polypeptides, or agonists or antagonists of D-SLAM, that
can decrease
hemostatic or thrombolytic activity could be used to inhibit or dissolve
clotting, important in
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the treatment, diagnosis, detection, and/or prevention of heart attacks
(infarction), strokes, or
scarring.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may also be useful in treating, diagnosing, detecting, and/or preventing
autoimmune
disorders. 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 D-
SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
that can
inhibit an immune response, particularly the proliferation, differentiation,
or chemotaxis of T-
cells, may be an effective therapy in preventing autoimmune disorders.
Examples of autoimmune disorders that can be treated, diagnosed, detected,
and/or
prevented include, but are not limited to: Addison's Disease, hemolytic
anemia,
antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic
encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple
Sclerosis,
Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,
Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,
Autoimmune
Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune
inflammatory
eye disease.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic
asthma) or other respiratory problems, may also be treated, diagnosed,
detected, andlor
prevented by D-SLAM polynucleotides or polypeptides, or agonists or
antagonists of D-
SLAM. Moreover, these molecules can be used to treat, diagnose, detect, and/or
prevent
anaphylaxis, hypersensitivity to an antigenic molecule, or blood group
incompatibility.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may also be used to treat, diagnose, detect, and/or prevent organ rejection or
graft-versus-host
disease (GVHD). 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 D-SLAM polynucleotides or polypeptides, or agonists or
antagonists of D-
SLAM, that inhibits an immune response, particularly the proliferation,
differentiation, or
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chemotaxis of T-cells, may be an effective therapy in preventing organ
rejection or GVHD.
Similarly, D-SLAM polynucleotides or polypeptides, or agonists or antagonists
of D-
SLAM, may also be used to modulate inflammation. For example, D-SLAM
polynucleotides
or polypeptides, or agonists or antagonists of D-SLAM, may inhibit the
proliferation and
differentiation of cells involved in an inflammatory response. These molecules
can be used
to treat, diagnose, detect, and/or prevent inflammatory conditions, both
chronic and acute
conditions, including inflammation associated with infection (e.g., septic
shock, sepsis, or
systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury,
endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or
resulting
from over production of cytokines (e.g., TNF or IL-1.)
Hyper~roliferative Disorders
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
can
be used to treat, diagnose, detect, and/or prevent hyperproliferative
disorders, including
neoplasms. D-SLAM polynucleotides or polypeptides, or agonists or antagonists
of D
SLAM, may inhibit the proliferation of the disorder through direct or indirect
interactions.
Alternatively, D-SLAM polynucleotides or polypeptides, or agonists or
antagonists of D
SLAM, may proliferate other cells which can inhibit the hyperproliferative
disorder.
For example, by increasing an immune response, particularly increasing
antigenic
qualities of the hyperproliferative disorder or by proliferating,
differentiating, or mobilizing
T-cells, hyperproliferative disorders can be treated, diagnosed, detected,
and/or prevented.
This immune response may be increased by either enhancing an existing immune
response, or
by initiating a new immune response. Alternatively, decreasing an immune
response may
also be a method of treating, diagnosing, detecting, and/or preventing
hyperproliferative
disorders, such as a chemotherapeutic agent.
Examples of hyperproliferative disorders that can be treated, diagnosed,
detected,
and/or prevented by D-SLAM polynucleotides or polypeptides, or agonists or
antagonists of
D-SLAM, include, but are not limited to neoplasms located in the: abdomen,
bone, breast,
digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid,
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pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous
(central and
peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic,
and urogenital.
Similarly, other hyperproliferative disorders can also be treated, diagnosed,
detected,
and/or prevented by D-SLAM polynucleotides or polypeptides, or agonists or
antagonists of
D-SLAM. Examples of such hyperproliferative disorders include, but are not
limited to:
hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,
purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's
Disease,
histiocytosis, and any other hyperproliferative disease, besides neoplasia,
located in an organ
system listed above.
Cardiovascular Disorders
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
encoding D-SLAM may be used to treat, diagnose, detect, and/or prevent
cardiovascular
disorders, including peripheral artery disease, such as limb ischemia.
Cardiovascular disorders include cardiovascular abnormalities, such as arterio-
arterial
fistula, arteriovenous fistula, cerebral arteriovenous malformations,
congenital heart defects,
pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include
aortic
coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart,
dextrocardia, patent
ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left
heart syndrome,
levocardia, tetralogy of fallot, transposition of great vessels, double outlet
right ventricle,
tricuspid atresia, persistent truncus arteriosus, and heart septal defects,
such as
aortopulmonary septal defect, endocardial cushion defects, Lutembacher's
Syndrome, trilogy
of Fallot, ventricular heart septal defects.
Cardiovascular disorders also include heart disease, such as arrhythmias,
carcinoid
heart disease, high cardiac output, low cardiac output, cardiac tamponade,
endocarditis
(including bacterial), heart aneurysm, cardiac arrest, congestive heart
failure, congestive
cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,
congestive
cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy,
post-infarction
heart rupture, ventricular septal rupture, heart valve diseases, myocardial
diseases, myocardial
ischemia, pericardial effusion, pericarditis (including constrictive and
tuberculous),
pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease,
rheumatic heart
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disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy
complications,
Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia,
extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block,
long QT
syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation
syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias,
and
ventricular fibrillation. Tachycardias include paroxysmal tachycardia,
supraventricular
tachycardia, accelerated idioventricular rhythm, atrioventricular nodal
reentry tachycardia,
ectopic atrial tachycardia, ectopic functional tachycardia, sinoatrial nodal
reentry tachycardia,
sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve disease include aortic valve insufficiency, aortic valve stenosis,
hear
murmurs, aortic valve prolapse, mifral valve prolapse, tricuspid valve
prolapse, mural valve
insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve
insufficiency,
pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency,
and tricuspid valve
stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy,
hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary
subvalvular stenosis,
restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and
myocarditis.
Myocardial ischemias include coronary disease, such as angina pectoris,
coronary
aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm,
myocardial
infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-
Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic
diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive
diseases, arteritis,
enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic
angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-
occlusive
disease, hypertension, hypotension, ischemia, peripheral vascular diseases,
phlebitis,
pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein
occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia,
atacia
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telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose
veins, varicose
ulcer, vasculitis, and venous insufficiency.
Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms,
ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms,
heart
aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent
claudication, carotid
stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya
disease, renal
artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
Cerebrovascular disorders include carotid artery diseases, cerebral amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformation, cerebral artery diseases, cerebral embolism and
thrombosis,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral
hemorrhage,
epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral
infarction,
cerebral ischemia (including transient), subclavian steal syndrome,
periventricular
leukomalacia, vascular headache, cluster headache, migraine, and
vertebrobasilar
insufficiency.
Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms,
blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.
Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein
occlusion,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
and'thrombophlebitis.
Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes,
anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and
peripheral
limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome,
Churg-Strauss
Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans,
hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous
vasculitis, and
Wegener's granulomatosis.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
are
especially effective for the treatment, diagnosis, detection, and/or
prevention of critical limb
ischemia and coronary disease. As shown in the Examples, administration of D-
SLAM
polynucleotides and polypeptides to an experimentally induced ischemia rabbit
hindlimb may
restore blood pressure ratio, blood flow, angiographic score, and capillary
density.
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D-SLAM polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous injection,
topical administration, catheter infusion, biolistic injectors, particle
accelerators, gelfoam
sponge depots, other commercially available depot materials, osmotic pumps,
oral or
suppositorial solid pharmaceutical formulations, decanting or topical
applications during
surgery, aerosol delivery. Such methods are known in the art. D-SLAM
polypeptides may be
administered as part of a pharmaceutical composition, described in more detail
below.
Methods of delivering D-SLAM polynucleotides are described in more detail
herein.
Anti-Angiogenesis Activity
The naturally occurring balance between endogenous stimulators and inhibitors
of
angiogenesis is one in which inhibitory influences predominate. Rastinejad et
al., Cell
56:345-355 (1989). In those rare instances in which neovascularization occurs
under normal
physiological conditions, such as wound healing, organ regeneration, embryonic
development, and female reproductive processes, angiogenesis is stringently
regulated and
spatially and temporally delimited. Under conditions of pathological
angiogenesis such as
that characterizing solid tumor growth, these regulatory controls fail.
Unregulated
angiogenesis becomes pathologic and sustains progression of many neoplastic
and non-
neoplastic diseases. A number of serious diseases are dominated by abnormal
neovascularization including solid tumor growth and metastases, arthritis,
some types of eye
disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-
634 (1991);
Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.
Microvasc. Res.
29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and
Weinhouse,
Academic Press, New York, pp. 175-203 ( 1985); Patz, Am. J. Opthalmol. 94:715-
743
( 1982); and Folkman et al., Science 221:719-725 ( 1983). In a number of
pathological
conditions, the process of angiogenesis contributes to the disease state. For
example,
significant data have accumulated which suggest that the growth of solid
tumors is dependent
on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
The present invention provides for treatment, diagnosis, detection, and/or
prevention
of diseases or disorders associated with neovascularization by administration
of the D-SLAM
polynucleotides and/or polypeptides of the invention, as well as agonists or
antagonists of D-
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SLAM. Malignant and metastatic conditions which can be treated, diagnosed,
detected,
and/or prevented with the polynucleotides and polypeptides, or agonists or
antagonists of the
invention include, but are not limited to, malignancies, solid tumors, and
cancers described
herein and otherwise known in the art (for a review of such disorders, see
Fishman et al.,
Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia ( 1985)):
Ocular disorders associated with neovascularization which can be treated,
diagnosed,
detected, and/or prevented with the D-SLAM polynucleotides and polypeptides of
the present
invention (including D-SLAM agonists and/or antagonists) include, but are not
limited to:
neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental
fibroplasia, uveitis,
retinopathy of prematurity macular degeneration, corneal graft
neovascularization, as well as
other eye inflammatory diseases, ocular tumors and diseases associated with
choroidal or iris
neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal.
85:704-710 (1978)
and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
Additionally, disorders which can be treated, diagnosed, detected, and/or
prevented
with the D-SLAM polynucleotides and polypeptides of the present invention
(including D-
SLAM agonist and/or antagonists) include, but are not limited to, hemangioma,
arthritis,
psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing,
granulations,
hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber
syndrome, pyogenic
granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, disorders and/or states, which can be treated, diagnosed, detected,
and/or
prevented with the D-SLAM polynucleotides and polypeptides of the present
invention
(including D-SLAM agonist and/or antagonists) include, but are not limited to,
solid tumors,
blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma,
benign tumors, for
example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for
example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration, corneal graft
rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and
uvietis, delayed
wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars
(keloids),
nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial
angiogenesis,
coronary collaterals, cerebral collaterals, arteriovenous malformations,
ischemic limb
angiogenesis, Osler-Webber Syndrome, plaque neovascularization,
telangiectasia,
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hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation,
Crohn's
disease, atherosclerosis, birth control agent by preventing vascularization
required for embryo
implantation controlling menstruation, diseases that have angiogenesis as a
pathologic
consequence such as cat scratch disease (Rochele minalia quintosa), ulcers
(Helicobacter
pylori), Bartonellosis and bacillary angiomatosis.
Diseases at the Cellular Level
Diseases associated with increased cell survival or the inhibition of
apoptosis that
could be treated, diagnosed, detected, and/or prevented by D-SLAM
polynucleotides or
polypeptides, as well as antagonists or agonists of D-SLAM, include cancers
(such as
follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent
tumors,
including, but not limited to colon cancer, cardiac tumors, pancreatic cancer,
melanoma,
retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular
cancer, stomach
cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma,
osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate
cancer,
Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple
sclerosis,
Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's
disease, Crohn's
disease, polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis
and rheumatoid arthritis) and viral infections (such as herpes viruses, pox
viruses and
adenoviruses), inflammation, graft v. host disease, acute graft rejection, and
chronic graft
rejection. In preferred embodiments, D-SLAM polynucleotides, polypeptides,
and/or
antagonists of the invention are used to inhibit growth, progression, and/or
metasis of
cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that
could be
treated, diagnosed, detected, and/or prevented by D-SLAM polynucleotides or
polypeptides,
or agonists or antagonists of D-SLAM, include, but are not limited to,
progression, and/or
metastases of malignancies and related disorders such as leukemia (including
acute leukemias
(e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic,
promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias
(e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia)),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's
disease), multiple
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myeloma, Waldenstrorri s macroglobulinemia, heavy chain disease, and solid
tumors
including, but not limited to, sarcomas and carcinomas such as 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, Wilm's 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, menangioma, melanoma,
neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated, diagnosed,
detected, and/or prevented by D-SLAM polynucleotides or polypeptides, as well
as agonists
or antagonists of D-SLAM, include AIDS; neurodegenerative disorders (such as
Alzheimer's
disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis
pigmentosa, Cerebellar
degeneration and brain tumor or prior associated disease); autoimmune
disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's
disease, Crohn's disease, polymyositis, systemic lupus erythematosus and
immune-related
glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such
as aplastic
anemia), graft v. host disease, ischemic injury (such as that caused by
myocardial infarction,
stroke and reperfusion injury), liver injury (e.g., hepatitis related liver
injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer);
toxin-induced
liver disease (such as that caused by alcohol), septic shock, cachexia and
anorexia.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is
provided a
process for utilizing D-SLAM polynucleotides or polypeptides, as well as
agonists or
antagonists of D-SLAM, for therapeutic purposes, for example, to stimulate
epithelial cell
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proliferation and basal keratinocytes for the purpose of wound healing, and to
stimulate hair
follicle production and healing of dermal wounds. D-SLAM polynucleotides or
polypeptides,
as well as agonists or antagonists of D-SLAM, may be clinically useful in
stimulating wound
healing including surgical wounds, excisional wounds, deep wounds involving
damage of the
dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity
wounds, diabetic
ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers,
burns resulting from
heat exposure or chemicals, and other abnormal wound healing conditions such
as uremia,
malnutrition, vitamin deficiencies and complications associted with systemic
treatment with
steroids, radiation therapy and antineoplastic drugs and antimetabolites. D-
SLAM
polynucleotides or polypeptides, as well as agonists or antagonists of D-SLAM,
could be
used to promote dermal reestablishment subsequent to dermal loss
D-SLAM polynucleotides or polypeptides, as well as agonists or antagonists of
D-
SLAM, could be used to increase the adherence of skin grafts to a wound bed
and to
stimulate re-epithelialization from the wound bed. The following are types of
grafts that D-
SLAM polynucleotides or polypeptides, agonists or antagonists of D-SLAM, could
be used
to increase adherence to a wound bed: autografts, artificial skin, allografts,
autodermic graft,
autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft,
brephoplastic grafts,
cubs graft, delayed graft, dermic graft, epidermis graft, fascia graft, full
thickness graft,
heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar
graft, mesh
graft, mucosal graft, Oilier-Thiersch graft, omenpal graft, patch graft,
pedicle graft,
penetrating graft, split skin graft, thick split graft. D-SLAM polynucleotides
or polypeptides,
as well as agonists or antagonists of D-SLAM, can be used to promote skin
strength and to
improve the appearance of aged skin.
It is believed that D-SLAM polynucleotides or polypeptides, as well as
agonists or
antagonists of D-SLAM, will also produce changes in hepatocyte proliferation,
and epithelial
cell proliferation in the lung, breast, pancreas, stomach, small intesting,
and large intestine.
D-SLAM polynucleotides or polypeptides, as well as agonists or antagonists of
D-SLAM,
could promote proliferation of epithelial cells such as sebocytes, hair
follicles, hepatocytes,
type II pneumocytes, mucin-producing goblet cells, and other epithelial cells
and their
progenitors contained within the skin, lung, liver, and gastrointestinal
tract. D-SLAM
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polynucleotides or polypeptides, agonists or antagonists of D-SLAM, may
promote
proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
D-SLAM polynucleotides or polypeptides, as well as agonists or antagonists of
D-
SLAM, could also be used to reduce the side effects of gut toxicity that
result from radiation,
chemotherapy treatments or viral infections. D-SLAM polynucleotides or
polypeptides, as
well as agonists or antagonists of D-SLAM, may have a cytoprotective effect on
the small
intestine mucosa. D-SLAM polynucleotides or polypeptides, as well as agonists
or
antagonists of D-SLAM, may also stimulate healing of mucositis (mouth ulcers)
that result
from chemotherapy and viral infections.
D-SLAM polynucleotides or polypeptides, as well as agonists or antagonists of
D-
SLAM, could further be used in full regeneration of skin in full and partial
thickness skin
defects, including burns, (i.e., repopulation of hair follicles, sweat glands,
and sebaceous
glands), treatment, diagnosis, detection, and/or prevention of other skin
defects such as
psoriasis. D-SLAM polynucleotides or polypeptides, as well as agonists or
antagonists of D-
SLAM, could be used to treat, diagnose, detect, and/or prevent epidermolysis
bullosa, a
defect in adherence of the epidermis to the underlying dermis which results in
frequent, open
and painful blisters by accelerating reepithelialization of these lesions. D-
SLAM
polynucleotides or polypeptides, as well as agonists or antagonists of D-SLAM,
could also be
used to treat, diagnose, detect, and/or prevent gastric and doudenal ulcers
and help heal by
scar formation of the mucosal lining and regeneration of glandular mucosa and
duodenal
mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's
disease and
ulcerative colitis, are diseases which result in destruction of the mucosal
surface of the small
or large intestine, respectively. Thus, D-SLAM polynucleotides or
polypeptides, as well as
agonists or antagonists of D-SLAM, could be used to promote the resurfacing of
the mucosal
surface to aid more rapid healing and to prevent progression of inflammatory
bowel disease.
Treatment, diagnosis, detection, and/or prevention with D-SLAM polynucleotides
or
polypeptides, agonists or antagonists of D-SLAM, is expected to have a
significant effect on
the production of mucus throughout the gastrointestinal tract and could be
used to protect the
intestinal mucosa from injurious substances that are ingested or following
surgery. D-SLAM
polynucleotides or polypeptides, as well as agonists or antagonists of D-SLAM,
could be
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used to treat, diagnose, detect, and/or prevent diseases associate with the
under expression of
D-SLAM.
Moreover, D-SLAM polynucleotides or polypeptides, as well as agonists or
antagonists of D-SLAM, could be used to prevent and heal damage to the lungs
due to
various pathological states. A growth factor such as D-SLAM polynucleotides or
polypeptides, as well as agonists or antagonists of D-SLAM, which could
stimulate
proliferation and differentiation and promote the repair of alveoli and
brochiolar epithelium
to prevent or treat, diagnose, detect, and/or prevent acute or chronic lung
damage. For
example, emphysema, which results in the progressive loss of aveoli, and
inhalation injuries,
i.e., resulting from smoke inhalation and burns, that cause necrosis of the
bronchiolar
epithelium and alveoli could be effectively treated, diagnosed, detected,
and/or prevented
using D-SLAM polynucleotides or polypeptides, agonists or antagonists of D-
SLAM. Also,
D-SLAM polynucleotides or polypeptides, as well as agonists or antagonists of
D-SLAM,
could be used to stimulate the proliferation of and differentiation of type II
pneumocytes,
which may help treat, diagnose, detect, and/or prevent disease such as hyaline
membrane
diseases, such as infant respiratory distress syndrome and bronchopulmonary
displasia, in
premature infants.
D-SLAM polynucleotides or polypeptides, as well as agonists or antagonists of
D
SLAM, could stimulate the proliferation and differentiation of hepatocytes
and, thus, could
be used to alleviate, treat, diagnose, detect, and/or prevent liver diseases
and pathologies such
as fulminant liver failure caused by cirrhosis, liver damage caused by viral
hepatitis and toxic
substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins
known in the
art).
In addition, D-SLAM polynucleotides or polypeptides, as well as agonists or
antagonists of D-SLAM, could be used treat, diagnose, detect, and/or prevent
the onset of
diabetes mellitus. In patients with newly diagnosed Types I and II diabetes,
where some islet
cell function remains, D-SLAM polynucleotides or polypeptides, as well as
agonists or
antagonists of D-SLAM, could be used to maintain the islet function so as to
alleviate, delay
or prevent permanent manifestation of the disease. Also, D-SLAM
polynucleotides or
polypeptides, as well as agonists or antagonists of D-SLAM, could be used as
an auxiliary in
islet cell transplantation to improve or promote islet cell function.
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Infectious Disease
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
can
be used to treat, diagnose, detect, and/or prevent infectious agents. For
example, by
increasing the immune response, particularly increasing the proliferation and
differentiation
of B and/or T cells, infectious diseases may be treated, diagnosed, detected,
and/or prevented.
The immune response may be increased by either enhancing an existing immune
response, or
by initiating a new immune response. ~ Alternatively, D-SLAM polynucleotides
or
polypeptides, or agonists or antagonists of D-SLAM, may also directly inhibit
the infectious
agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms
that can be treated, diagnosed, detected, and/or prevented by D-SLAM
polynucleotides or
polypeptides, or agonists or antagonists of D-SLAM. Examples of viruses,
include, but are
not limited to the following DNA and RNA viral families: Arbovirus,
Adenoviridae,
Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,
Circoviridae,
Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such
as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,
Paramyxoviridae,
Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza),
Papovaviridae,
Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),
Reoviridae (e.g.,
Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,
Rubivirus).
Viruses falling within these families can cause a variety of diseases or
symptoms, including,
but not limited to: arthritis, bronchiollitis, encephalitis, eye infections
(e.g., conjunctivitis,
keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active,
Delta), meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma,
chickenpox ,
hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold,
Polio,
leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g.,
Kaposi's, warts), and
viremia. D-SLAM polynucleotides or polypeptides, or agonists or antagonists of
D-SLAM,
can be used to treat, diagnose, detect, and/or prevent any of these symptoms
or diseases.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that can
be treated, diagnosed, detected, and/or prevented by D-SLAM polynucleotides or
polypept'ides, or agonists or antagonists of D-SLAM, include, but not limited
to, the
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following Gram-Negative and Gram-positive bacterial families and fungi:
Actinomycetales
(e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae
(e.g.,
Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia,
Brucellosis,
Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,
Dermatocycoses,
Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia),
Erysipelothrix, Helicobacter,
Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g.,
Acinetobacter,
Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g., Actinobacillus,
Heamophilus,
Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and
Staphylococcal.
These bacterial or fungal families can cause the following diseases or
symptoms, including,
but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis,
uveitis), gingivitis, opportunistic infections (e.g., AIDS related
infections), paronychia,
prosthesis-related infections, Reiter's Disease, respiratory tract infections,
such as Whooping
Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,
Paratyphoid
Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia,
Syphilis,
Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism,
gangrene, tetanus,
impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g.,
cellulitis, dermatocycoses), toxemia, urinary tract infections, wound
infections. D-SLAM
polynucleotides or polypeptides, or agonists or antagonists of D-SLAM, can be
used to treat,
diagnose, detect, and/or prevent any of these symptoms or diseases.
Moreover, parasitic agents causing disease or symptoms that can be treated,
diagnosed, detected, and/or prevented by D-SLAM polynucleotides or
polypeptides, or
agonists or antagonists of D-SLAM, include, but not limited to, the following
families:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,
Dourine,
Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,
Toxoplasmosis,
Trypanosomiasis, and Trichomonas. These parasites can cause a variety of
diseases or
symptoms, including, but not limited to: Scabies, Trombiculiasis, eye
infections, intestinal
disease (e.g., dysentery, giardiasis), liver disease, lung disease,
opportunistic infections (e.g.,
AIDS related), Malaria, pregnancy complications, and toxoplasmosis. D-SLAM
polynucleotides or polypeptides, or agonists or antagonists of D-SLAM, can be
used to treat,
diagnose, detect, and/or prevent any of these symptoms or diseases.
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Preferably, treatment, diagnosis, detection, and/or prevention using D-SLAM
polynucleotides or polypeptides, or agonists or antagonists of D-SLAM, could
either be by
administering an effective amount of D-SLAM polypeptide to the patient, or by
removing
cells from the patient, supplying the cells with D-SLAM polynucleotide, and
returning the
engineered cells to the patient (ex vivo therapy). Moreover, the D-SLAM
polypeptide or
polynucleotide can be used as an antigen in a vaccine to raise an immune
response against
infectious disease.
Regeneration
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
can
be used to differentiate, proliferate, and attract cells, leading to the
regeneration of tissues.
(See, Science 276:59-87 (1997).) The regeneration of tissues could be used to
repair, replace,
or protect tissue damaged by congenital defects, trauma (wounds, burns,
incisions, or ulcers),
age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver
failure), surgery,
including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic
cytokine damage.
Tissues that could be regenerated using the present invention include organs
(e.g.,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous, hematopoietic, and
skeletal (bone,
cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs
without or decreased
scarring. Regeneration also may include angiogenesis.
Moreover, D-SLAM polynucleotides or polypeptides, or agonists or antagonists
of D-
SLAM, may increase regeneration of tissues difficult to heal. For example,
increased
tendon/ligament regeneration would quicken recovery time after damage. D-SLAM
polynucleotides or polypeptides, or agonists or antagonists of D-SLAM, of the
present
invention could also be used prophylactically in an effort to avoid damage.
Specific diseases
that could be treated, diagnosed, detected, and/or prevented include of
tendinitis, carpal
tunnel syndrome, and other tendon or ligament defects. A further example of
tissue
regeneration of non-healing wounds includes pressure ulcers, ulcers associated
with vascular
insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using D-SLAM
polynucleotides or polypeptides, or agonists or antagonists of D-SLAM, to
proliferate and
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differentiate nerve cells. Diseases that could be treated, diagnosed,
detected, and/or
prevented using this method include central and peripheral nervous system
diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal cord
disorders, head trauma,
cerebrovascular disease, and stoke). Specifically, diseases associated with
peripheral nerve
injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other
medical
therapies), localized neuropathies, and central nervous system diseases (e.g.,
Alzheimer's
disease, Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis, and Shy-
Drager syndrome), could all be treated, diagnosed, detected, and/or prevented
using the D-
SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM.
Chemotaxis
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes
cells (e.g.,
monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,
epithelial and/or
endothelial cells) to a particular site in the body, such as inflammation,
infection, or site of
hyperproliferation. The mobilized cells can then fight off and/or heal the
particular trauma or
abnormality.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may increase chemotaxic activity of particular cells. These chemotactic
molecules can then
be used to treat, diagnose, detect, and/or prevent inflammation, infection,
hyperproliferative
disorders, or any immune system disorder by increasing the number of cells
targeted to a
particular location in the body. For example, chemotaxic molecules can be used
to treat,
diagnose, detect, and/or prevent wounds and other trauma to tissues by
attracting immune
cells to the injured location. As a chemotactic molecule, D-SLAM could also
attract
fibroblasts, which can be used to treat, diagnose, detect, and/or prevent
wounds.
It is also contemplated that D-SLAM polynucleotides or polypeptides, or
agonists or
antagonists of D-SLAM, may inhibit chemotactic activity. These molecules could
also be
used to treat, diagnose, detect, and/or prevent disorders. Thus, D-SLAM
polynucleotides or
polypeptides, or agonists or antagonists of D-SLAM, could be used as an
inhibitor of
chemotaxis.
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Binding Activity
D-SLAM polypeptides may be used to screen for molecules that bind to D-SLAM or
for molecules to which D-SLAM binds. The binding of D-SLAM and the molecule
may
activate (agonist), increase, inhibit (antagonist), or decrease activity of
the D-SLAM or the
molecule bound. Examples of such molecules include antibodies,
oligonucleotides, proteins
(e.g., receptors),or small molecules.
Preferably, the molecule is closely related to the natural ligand of D-SLAM,
e.g., a
fragment of the ligand, or a natural substrate, a ligand, a structural or
functional mimetic.
(See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)
Similarly, the
molecule can be closely related to the natural receptor to which D-SLAM binds,
or at least, a
fragment of the receptor capable of being bound by D-SLAM (e.g., active site).
In either
case, the molecule can be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate
cells
which express D-SLAM, either as a secreted protein or on the cell membrane.
Preferred cells
include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing D-
SLAM(or cell
membrane containing the expressed polypeptide) are then preferably contacted
with a test
compound potentially containing the molecule to observe binding, stimulation,
or inhibition
of activity of either D-SLAM or the molecule.
The assay may simply test binding of a candidate compound toD-SLAM, wherein
binding is detected by a label, or in an assay involving competition with a
labeled competitor.
Further, the assay may test whether the candidate compound results in a signal
generated by
binding to D-SLAM.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
mixtures. The assay may also simply comprise the steps of mixing a candidate
compound
with a solution containing D-SLAM, measuring D-SLAM/molecule activity or
binding, and
comparing the D-SLAM/molecule activity or binding to a standard.
Preferably, an ELISA assay can measure D-SLAM level or activity in a sample
(e.g.,
biological sample) using a monoclonal or polyclonal antibody. The antibody can
measure D-
SLAM level or activity by either binding, directly or indirectly, to D-SLAM or
by competing
with D-SLAM for a substrate.
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Additionally, the receptor to which D-SLAM binds can be identified by numerous
methods known to those of skill in the art, for example, ligand panning and
FACS sorting
(Coligan, et al., Current Protocols in Immun., 1 (2), Chapter 5, ( 1991 )).
For example,
expression cloning is employed wherein polyadenylated RNA is prepared from a
cell
responsive to the polypeptides, for example, NIH3T3 cells which are known to
contain
multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA
library created
from this RNA is divided into pools and used to transfect COS cells or other
cells that are not
responsive to the polypeptides. Transfected cells which are grown on glass
slides are
exposed to the polypeptide of the present invention, after they have been
labelled. The
polypeptides can be labeled by a variety of means including iodination or
inclusion of a
recognition site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-
radiographic
analysis. Positive pools are identified and sub-pools are prepared and re-
transfected using an
iterative sub-pooling and re-screening process, eventually yielding a single
clones that
encodes the putative receptor.
As an alternative approach for receptor identification, the labeled
polypeptides can be
photoaffinity linked with cell membrane or extract preparations that express
the receptor
molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-
ray film.
The labeled complex containing the receptors of the polypeptides can be
excised, resolved
into peptide fragments, and subjected to protein microsequencing. The amino
acid sequence
obtained from microsequencing would be used to design a set of degenerate
oligonucleotide
probes to screen a cDNA library to identify the genes encoding the putative
receptors.
Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling,
and/or
codon-shuffling (collectively referred to as "DNA shuffling") may be employed
to modulate
the activities of D-SLAM thereby effectively generating agonists and
antagonists of D
SLAM. See generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721,
5,834,252, and
5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechreol. 8:724-33
(1997); Harayama, S.
Trends Biotechnol. 16(2):76-82 ( 1998); Hansson, L. O., et al., J. Mol. Biol.
287:265-76
(1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998)
(each of
these patents and publications are hereby incorporated by reference). In one
embodiment,
alteration of D-SLAM polynucleotides and corresponding polypeptides may be
achieved by
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DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments
into a
desired D-SLAM molecule by homologous, or site-specific, recombination. In
another
embodiment, D-SLAM polynucleotides and corresponding polypeptides may be
alterred 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
components,
motifs, sections, parts, domains, fragments, etc., of D-SLAM may be 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 Secreted
Lymphocyte Activation Molecule (SLAM) family members. In further preferred
embodiments, the heterologous molecule is a growth factor such as, for
example,
platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I),
transforming
growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth
factor (FGF),
TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7,
activins
A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation
factors (GDFs),
nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-betas, and
glial-derived
neurotrophic factor (GDNF).
Other preferred fragments are biologically active D-SLAM fragments.
Biologically
active fragments are those exhibiting activity similar, but not necessarily
identical, to an
activity of the D-SLAM polypeptide. The biological activity of the fragments
may include an
improved desired activity, or a decreased undesirable activity.
Additionally, this invention provides a method of screening compounds to
identify
those which modulate the action of the polypeptide of the present invention.
An example of
such an assay comprises combining a mammalian fibroblast cell, a the
polypeptide of the
present invention, the compound to be screened and ['H] thymidine under cell
culture
conditions where the fibroblast cell would normally proliferate. A control
assay may be
performed in the absence of the compound to be screened and compared to the
amount of
fibroblast proliferation in the presence of the compound to determine if the
compound
stimulates proliferation by determining the uptake of [3H] thymidine in each
case. The
amount of fibroblast cell proliferation is measured by liquid scintillation
chromatography
which measures the incorporation of [3H] thymidine. Both agonist and
antagonist compounds
may be identified by this procedure.
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In another method, a mammalian cell or membrane preparation expressing a
receptor
for a polypeptide of the present invention is incubated with a labeled
polypeptide of the
present invention in the presence of the compound. The ability of the compound
to enhance
or block this interaction could then be measured. Alternatively, the response
of a known
second messenger system following interaction of a compound to be screened and
the D-
SLAM receptor is measured and the ability of the compound to bind to the
receptor and elicit
a second messenger response is measured to determine if the compound is a
potential agonist
or antagonist. Such second messenger systems include but are not limited to,
CAMP
guanylate cyclase, ion channels or phosphoinositide hydrolysis.
All of these above assays can be used as diagnostic or prognostic markers. The
molecules discovered using these assays can be used to treat, diagnose,
detect, and/or prevent
disease or to bring about a particular result in a patient (e.g., blood vessel
growth) by
activating or inhibiting the D-SLAM/molecule. Moreover, the assays can
discover agents
which may inhibit or enhance the production of D-SLAM from suitably
manipulated cells or
tissues.Therefore, the invention includes a method of identifying compounds
which bind to
D-SLAM comprising the steps of: (a) incubating a candidate binding compound
with D
SLAM; and (b) determining if binding has occurred. Moreover, the invention
includes a
method of identifying agonists/antagonists comprising the steps of: (a)
incubating a
candidate compound with D-SLAM, (b) assaying a biological activity , and (b)
determining if
a biological activity of D-SLAM has been altered.
Also, one could identify molecules bind D-SLAM experimentally by using the
beta-
pleated sheet regions disclosed in Figure 3 and Table 1. Accordingly, specific
embodiments
of the invention are directed to polynucleotides encoding polypeptides which
comprise, or
alternatively consist of, the amino acid sequence of each beta pleated sheet
regions disclosed
in Figure 3/Table 1. Additional embodiments of the invention are directed to
polynucleotides encoding D-SLAM polypeptides which comprise, or alternatively
consist of,
any combination or all of the beta pleated sheet regions disclosed in Figure
3/Table 1.
Additional preferred embodiments of the invention are directed to polypeptides
which
comprise, or alternatively consist of, the D-SLAM amino acid sequence of each
of the beta
pleated sheet regions disclosed in Figure 3/Table 1. Additional embodiments of
the invention
are directed to D-SLAM polypeptides which comprise, or alternatively consist
of, any
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combination or all of the beta pleated sheet regions disclosed in Figure
3/Table 1.
Antisense And Ribozyme (Anta;. one fists)
In specific embodiments, antagonists according to the present invention are
nucleic
acids corresponding to the sequences contained in SEQ ID NO:1, or the
complementary
strand thereof, and/or to nucleotide sequences contained in the deposited
clone 209623. In
one embodiment, antisense sequence is generated internally by the organism, in
another
embodiment, the antisense sequence is separately administered (see, for
example, O'Connor,
J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of
Gene
Expression, CRC Press, Boca Raton, FL (1988). Antisense technology can be used
to control
gene expression through antisense DNA or RNA, or through triple-helix
formation.
Antisense techniques are discussed for example, in Okano, J., Neurochem.
56:560 ( 1991 );
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press,
Boca Raton,
FL ( 1988). Triple helix formation is discussed in, for instance, Lee et al.,
Nucleic Acids
Research 6:3073 ( 1979); Cooney et al., Science 241:456 ( 1988); and Dervan et
al., Science
251:1300 ( 1991 ). The methods are based on binding of a polynucleotide to a
complementary
DNA or RNA.
For example, the 5' coding portion of a polynucleotide that encodes the mature
polypeptide of the present invention may be used to design an antisense RNA
oligonucleotide
of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed
to be
complementary to a region of the gene involved in transcription thereby
preventing
transcription and the production of the receptor. The antisense RNA
oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into receptor
polypeptide.
In one embodiment, the D-SLAM antisense nucleic acid of the invention is
produced
intracellularly by transcription from an exogenous sequence. For example, a
vector or a
portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of
the invention.
Such a vector would contain a sequence encoding the D-SLAM antisense nucleic
acid. Such
a vector can remain episomal or become chromosomally integrated, as long as it
can be
transcribed to produce the desired antisense RNA. Such vectors can be
constructed by
recombinant DNA technology methods standard in the art. Vectors can be
plasmid, viral, or
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others know in the art, used for replication and expression in vertebrate
cells. Expression of
the sequence encoding D-SLAM, or fragments thereof, can be by any promoter
known in the
art to act in vertebrate, preferably human cells. Such promoters can be
inducible or
constitutive. Such promoters include, but are not limited to, the SV40 early
promoter region
(Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the
3' long
terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (
1980), the herpes
thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445
(1981), the
regulatory sequences of the metallothionein gene (Brinster, et al., Nature
296:39-42 (1982)),
etc.
The antisense nucleic acids of the invention comprise, or alternatively
consist of, a
sequence complementary to at least a portion of an RNA transcript of a D-SLAM
gene.
However, absolute complementarity, although preferred, is not required. A
sequence
"complementary to at least a portion of an RNA," referred to herein, means a
sequence
having sufficient complementarity to be able to hybridize with the RNA,
forming a stable
duplex; in the case of double stranded D-SLAM antisense nucleic acids, a
single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed. The
ability to
hybridize will depend on both the~of complementarity and the length of the
antisense nucleic
acid Generally, the larger the hybridizing nucleic acid, the more base
mismatches with a D-
SLAM RNA it may contain and still form a stable duplex (or triplex as the case
may be).
One skilled in the art can ascertain a tolerable~of mismatch by use of
standard procedures to
determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5' end of the message, e.g.,
the 5'
untranslated sequence up to and including the AUG initiation codon, should
work most
efficiently at inhibiting translation. However, sequences complementary to the
3'
untranslated sequences of mRNAs have been shown to be effective at inhibiting
translation of
mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335. Thus,
oligonucleotides complementary to either the 5'- or 3'- non- translated, non-
coding regions of
D-SLAM shown in Figures 1 A-B could be used in an antisense approach to
inhibit translation
of endogenous D-SLAM mRNA. Oligonucleotides complementary to the 5'
untranslated
region of the mRNA should include the complement of the AUG start codon.
Antisense
oligonucleotides complementary to mRNA coding regions are less efficient
inhibitors of
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translation but could be used in accordance with the invention. Whether
designed to
hybridize to the 5'-, 3'- or coding region of D-SLAM mRNA, antisense nucleic
acids should
be at least six nucleotides in length, and are preferably oligonucleotides
ranging from 6 to
about 50 nucleotides in length. In specific aspects the oligonucleotide is at
least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or double-stranded.
The
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate
backbone, for
example, to improve stability of the molecule, hybridization, etc. The
oligonucleotide may
include other appended groups such as peptides (e.g., for targeting host cell
receptors in
vivo), or agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al.,
1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.
Natl. Acad.
Sci. 84:648-652; PCT Publication No. W088/09810, published December 15, 1988)
or the
blood-brain barrier (see, e.g., PCT Publication No. W089/10134, published
April 25, 1988),
hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988,
BioTechniques 6:958-
976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549).
To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization
triggered cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety
which
is selected from the group including, but not limited to, 5-fluorouracil, 5-
bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-
D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-
methylthio-N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-
5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil, 3-(3-amino-
3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
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The antisense oligonucleotide may also comprise at least one modified sugar
moiety
selected from the group including, but not limited to, arabinose, 2-
fluoroarabinose, xylulose,
and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least
one
modified phosphate backbone selected from the group including, but not limited
to, a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a
formacetal or
analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
hybrids with
complementary RNA in which, contrary to the usual b-units, the strands run
parallel to each
other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The
oligonucleotide is a 2'-0-
methylribonucleotide (moue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a
chimeric
RNA-DNA analogue (moue et al., 1987, FEBS Lett. 215:327-330).
Polynucleotides of the invention may be synthesized by standard methods known
in
the art, e.g. by use of an automated DNA synthesizer (such as are commercially
available
from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides
may be synthesized by the method of Stein et al. ( 1988, Nucl. Acids Res.
16:3209),
methylphosphonate oligonucleotides can be prepared by use of controlled pore
glass polymer
supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451),
etc.
While antisense nucleotides complementary to the D-SLAM coding region sequence
could be used, those complementary to the transcribed untranslated region are
most preferred.
Potential antagonists according to the invention also include catalytic RNA,
or a
ribozyme (See, e.g., PCT International Publication WO 90/11364, published
October 4, 1990;
Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA
at site
specific recognition sequences can be used to destroy D-SLAM mRNAs, the use of
hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at
locations
dictated by flanking regions that form complementary base pairs with the
target mRNA. The
sole requirement is that the target mRNA have the following sequence of two
bases: 5'-UG-
3'. The construction and production of hammerhead ribozymes is well known in
the art and
is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).
There are
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numerous potential hammerhead ribozyme cleavage sites within the nucleotide
sequence of
D-SLAM (Figures lA-B). Preferably, the ribozyme is engineered so that the
cleavage
recognition site is located near the S' end of the D-SLAM mRNA; i.e., to
increase efficiency
and minimize the intracellular accumulation of non-functional mRNA
transcripts.
As in the antisense approach, the ribozymes of the invention can be composed
of
modified oligonucleotides (e.g_ for improved stability, targeting, etc.) and
should be delivered
to cells which express D-SLAM in vivo. DNA constructs encoding the ribozyme
may be
introduced into the cell in the same manner as described above for the
introduction of
antisense encoding DNA. A preferred method of delivery involves using a DNA
construct
"encoding" the ribozyme under the control of a strong constitutive promoter,
such as, for
example, pol III or pol II promoter, so that transfected cells will produce
sufficient quantities
of the ribozyme to destroy endogenous D-SLAM messages and inhibit translation.
Since
ribozymes unlike antisense molecules, are catalytic, a lower intracellular
concentration is
required for efficiency.
Antagonistlagonist compounds may be employed to inhibit the cell growth and
proliferation effects of the polypeptides of the present invention on
neoplastic cells and
tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or
prevent abnormal
cellular growth and proliferation, for example, in tumor formation or growth.
The antagonist/agonist may also be employed to prevent hyper-vascular
diseases, and
prevent the proliferation of epithelial lens cells after extracapsular
cataract surgery.
Prevention of the mitogenic activity of the polypeptides of the present
invention may also be
desirous in cases such as restenosis after balloon angioplasty.
The antagonist/agonist may also be employed to prevent the growth of scar
tissue
during wound healing.
The antagonist/agonist may also be employed to treat, diagnose, detect, and/or
prevent
the diseases described herein.
Other Activities
The polypeptide of the present invention, as a result of the ability to
stimulate
vascular endothelial cell growth, may be employed in treatment, diagnosis,
detection, and/or
prevention for stimulating re-vascularization of ischemic tissues due to
various disease
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conditions such as thrombosis, arteriosclerosis, and other cardiovascular
conditions. These
polypeptide may also be employed to stimulate angiogenesis and limb
regeneration, as
discussed above.
The polypeptide may also be employed for treating, diagnosing, detecting,
and/or
preventing wounds due to injuries, burns, post-operative tissue repair, and
ulcers since they
are mitogenic to various cells of different origins, such as fibroblast cells
and skeletal muscle
cells, and therefore, facilitate the repair or replacement of damaged or
diseased tissue.
The polypeptide of the present invention may also be employed stimulate
neuronal
growth and to treat, diagnose, detect, and/or prevent neuronal damage which
occurs in certain
neuronal disorders or neuro-degenerative conditions such as Alzheimer's
disease, Parkinson's
disease, and AIDS-related complex. D-SLAM may have the ability to stimulate
chondrocyte
growth, therefore, they may be employed to enhance bone and periodontal
regeneration and
aid in tissue transplants or bone grafts.
The polypeptide of the present invention may be also be employed to prevent
skin
aging due to sunburn by stimulating keratinocyte growth.
The D-SLAM polypeptide may also be employed for preventing hair loss, since
FGF
family members activate hair-forming cells and promotes melanocyte growth.
Along the
same lines, the polypeptides of the present invention may be employed to
stimulate growth
and differentiation of hematopoietic cells and bone marrow cells when used in
combination
with other cytokines.
The D-SLAM polypeptide may also be employed to maintain organs before
transplantation or for supporting cell culture of primary tissues.
The polypeptide of the present invention may also be employed for inducing
tissue of
mesodermal origin to differentiate in early embryos.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may also increase or decrease the differentiation or proliferation of
embryonic stem cells,
besides, as discussed above, hematopoietic lineage.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may also be used to modulate mammalian characteristics, such as body height,
weight, hair
color, eye color, skin, percentage of adipose tissue, pigmentation, size, and
shape (e.g.,
cosmetic surgery). Similarly, D-SLAM polynucleotides or polypeptides, or
agonists or
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antagonists of D-SLAM, may be used to modulate mammalian metabolism affecting
catabolism, anabolism, processing, utilization, and storage of energy.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may be used to change a mammal's mental state or physical state by influencing
biorhythms,
caricadic rhythms, depression (including depressive disorders), tendency for
violence,
tolerance for pain, reproductive capabilities (preferably by Activin or
Inhibin-like activity),
hormonal or endocrine levels, appetite, libido, memory, stress, or other
cognitive qualities.
D-SLAM polynucleotides or polypeptides, or agonists or antagonists of D-SLAM,
may also be used as a food additive or preservative, such as to increase or
decrease storage
capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals,
cofactors or other
nutritional components.
The above-recited applications have uses in a wide variety of hosts. Such
hosts
include, but are not limited to, human, murine, rabbit, goat, guinea pig,
camel, horse, mouse,
rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human
primate, and
human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig,
chicken, rat,
hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a
mammal. In most
preferred embodiments, the host is a human.
Having generally described the invention, the same will be more readily
understood
by reference to the following examples, which are provided by way of
illustration and are not
intended as limiting.
Examples
Example l: Isolation of the D-SLAM cDNA Clone From the Deposited Sample
The cDNA for D-SLAM is inserted into the SaII/NotI multiple cloning site of
pCMVSport 3Ø (Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD
20897.)
pCMVSport 3.0 contains an ampicillin resistance gene and may be transformed
into E. coli
strain DH10B, also available from Life Technologies. (See, for instance,
Gruber, C. E., et al.,
Focus I5: 59- ( 1993).)
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Two approaches can be used to isolate D-SLAM from the deposited sample. First,
a
specific polynucleotide of SEQ ID NO:1 with 30-40 nucleotides is synthesized
using an
Applied Biosystems DNA synthesizer according to the sequence reported. The
oligonucleotide is labeled, for instance, with 3zP-y ATP using T4
polynucleotide kinase and
purified according to routine methods. (E.g., Maniatis et al., Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY (1982).) The
plasmid
mixture is transformed into a suitable host (such as XL-1 Blue (Stratagene))
using techniques
known to those of skill in the art, such as those provided by the vector
supplier or in related
publications or patents. The transformants are plated on 1.5% agar plates
(containing the
appropriate selection agent, e.g., ampicillin) to a density of about 150
transformants
(colonies) per plate. These plates are screened using Nylon membranes
according to routine
methods for bacterial colony screening (e.g., Sambrook et al., Molecular
Cloning: A
Laboratory Manual, 2nd Edit., ( 1989), Cold Spring Harbor Laboratory Press,
pages 1.93 to
1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the
SEQ ID
NO:1 (i.e., within the region of SEQ ID NO:1 bounded by the 5' NT and the 3'
NT of the
clone) are synthesized and used to amplify the D-SLAM cDNA using the deposited
cDNA
plasmid as a template. The polymerase chain reaction is carried out under
routine conditions,
for instance, in 25 p1 of reaction mixture with 0.5 ug of the above cDNA
template. A
convenient reaction mixture is 1.5-5 mM MgCI" 0.01 % (w/v) gelatin, 20 NM each
of dATP,
dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
Thirty five
cycles of PCR (denaturation at 94~C for 1 min; annealing at 55~C for 1 min;
elongation at
72~C for 1 min) are performed with a Perkin-Elmer Cetus automated thermal
cycler. The
amplified product is analyzed by agarose gel electrophoresis and the DNA band
with
expected molecular weight is excised and purified. The PCR product is verified
to be the
selected sequence by subcloning and sequencing the DNA product.
Several methods are available for the identification of the 5' or 3' non-
coding portions
of the D-SLAM gene which may not be present in the deposited clone. These
methods
include but are not limited to, filter probing, clone enrichment using
specific probes, and
protocols similar or identical to 5' and 3' "RACE" protocols which are well
known in the art.
For instance, a method similar to 5' RACE is available for generating the
missing 5' end of a
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desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res.
21(7):1683-1684
( 1993).)
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a
population of
RNA presumably containing full-length gene RNA transcripts. A primer set
containing a
primer specific to the ligated RNA oligonucleotide and a primer specific to a
known sequence
of the D-SLAM gene of interest is used to PCR amplify the 5' portion of the D-
SLAM full-
length gene. This amplified product may then be sequenced and used to generate
the full
length gene.
This above method starts with total RNA isolated from the desired source,
although
poly-A+ RNA can be used. The RNA preparation can then be treated with
phosphatase if
necessary to eliminate 5' phosphate groups on degraded or damaged RNA which
may
interfere with the later RNA ligase step. The phosphatase should then be
inactivated and the
RNA treated with tobacco acid pyrophosphatase in order to remove the cap
structure present
at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at
the 5' end of
the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using
T4 RNA
ligase.
This modified RNA preparation is used as a template for first strand cDNA
synthesis
using a gene specific oligonucleotide. The first strand synthesis reaction is
used as a template
for PCR amplification of the desired 5' end using a primer specific to the
ligated RNA
oligonucleotide and a primer specific to the known sequence of the gene of
interest. The
resultant product is then sequenced and analyzed to confirm that the 5' end
sequence belongs
to the D-SLAM gene.
Example 2: Isolation of D-SLAM Genomic Clones
A human genomic Pl library (Genomic Systems, Inc.) is screened by PCR using
primers selected for the cDNA sequence corresponding to SEQ ID NO:1.,
according to the
method described in Example 1. (See also, Sambrook.)
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Example 3: Tissue Distribution of D-SLAM Polypeptides
Tissue distribution of mRNA expression of D-SLAM is determined using protocols
for Northern blot analysis, described by, among others, Sambrook et al. For
example, a D-
SLAM probe produced by the method described in Example 1 is labeled with P32
using the
rediprimeTM DNA labeling system (Amersham Life Science), according to
manufacturer's
instructions. After labeling, the probe is purified using CHROMA SPIN-100TM
column
(Clontech Laboratories, Inc.), according to manufacturer's protocol number
PT1200-1. The
purified labeled probe is then used to examine various human tissues for mRNA
expression.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or
human immune system tissues (IM) (Clontech) are examined with the labeled
probe using
ExpressHybTM hybridization solution (Clontech) according to manufacturer's
protocol
number PT1190-1. Following hybridization and washing, the blots are mounted
and exposed
to film at -70~C overnight, and the films developed according to standard
procedures.
Example 4: Chromosomal Mapping of D-SLAM
An oligonucleotide primer set is designed according to the sequence at the 5'
end of
SEQ ID NO:1. This primer preferably spans about 100 nucleotides. This primer
set is then
used in a polymerise chain reaction under the following set of conditions : 30
seconds, 95~C;
1 minute, 56~C; 1 minute, 70~C. This cycle is repeated 32 times followed by
one 5 minute
cycle at 70~C. Human, mouse, and hamster DNA is used as template in addition
to a somatic
cell hybrid panel containing individual chromosomes or chromosome fragments
(Bios, Inc).
The reactions is analyzed on either 8% polyacrylamide gels or 3.5 % agarose
gels.
Chromosome mapping is determined by the presence of an approximately 100 by
PCR
fragment in the particular somatic cell hybrid.
Example 5: Bacterial Expression of D-SLAM
D-SLAM polynucleotide encoding a D-SLAM polypeptide invention is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the
DNA sequence,
as outlined in Example l, to synthesize insertion fragments. The primers used
to amplify the
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cDNA insert should preferably contain restriction sites, such as BamHI and
XbaI, at the 5'
end of the primers in order to clone the amplified product into the expression
vector. For
example, BamHI and XbaI correspond to the restriction enzyme sites on the
bacterial
expression vector pQE-9. (Qiagen, Inc., Chatsworth, CA). This plasmid vector
encodes
S antibiotic resistance (Ampr), a bacterial origin of replication (ori), an
IPTG-regulatable
promoter/operator (P/0), a ribosome binding site (RBS), a 6-histidine tag (6-
His), and
restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is
ligated into the pQE-9 vector maintaining the reading frame initiated at the
bacterial RBS.
The ligation mixture is then used to transform the E. coli strain M15/rep4
(Qiagen, Inc.)
which contains multiple copies of the plasmid pREP4, which expresses the lacI
repressor and
also confers kanamycin resistance (Kanr). Transformants are identified by
their ability to
grow on LB plates and ampicillin/kanamycin resistant colonies are selected.
Plasmid DNA is
isolated and confirmed by restriction analysis.
Alternatively, a construct containing DNA encoding amino acid Q24-D233 of SEQ
ID N0:2 can be inserted into pQE70. This construct places a HIS tag (6
histidines) at the C-
terminus of the predicted extracellular domain of D-SLAM. Primers that can be
used include
a 5' primer containing a Sph restriction site, shown in bold:
GCAGCAGCATGCAAGTGCTGAGCAAAGTCGGGGGCTCGGTGCTG (SEQ ID NO:
14) and a 3' primer, containing a BgIII restriction site, shown in bold:
GCAGCAAGATCTATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:
15). This construct uses an ATG as a start codon contained within the SphI
site, then reading
into Q24 of SEQ ID N0:2, and continues until D233 of SEQ ID N0:2. The amino
acid
sequence encoded by this construct is as follows:
MQVLSKVGGSVLLVAARPPGFQVREAIWRSLWPSEELLATFFRGSLETLYHSRFLGR
AQLHSNLSLELGPLESGDSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIA
VERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPG
DRDVAYSCIVSNPVSWDLATVTPWDSCHHEAAPGKASYKDQVLSKVGGSVLLVAA
RPPGFQVREAIWRSLWPSEELLATFFRGSLETLYHSRFLGRAQLHSNLSLELGPLESG
DSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIAVERDAQPSKTCQVFLSC
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WAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPGDRDVAYSCIVSNPVSW
DLATVTPWDSCHHEAAPGKASYKDHHHHHH (SEQ ID NO: 16).
Alternatively, a His tag can be placed on the N-terminus of the predicted
mature form
containing only the extracellular domain of D-SLAM (e.g., corresponding to A23-
D233 of
SEQ ID N0:2). In this example, the 5' primer, containing a BamHI restriction
site, indicated
in bold, can be used:
GCAGCAGGATCCGCCCAAGTGCTGAGCAAAGTCGGGGGCTCGGTG (SEQ ID NO:
17) and a 3' primer, containing a HindIII restriction site, indicated in bold,
can be used:
GCAGCAAAGCTTTTAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID
NO: 18). These primer can be used to amplify DNA encoding A23-D233, and then
the
generated product can be inserted into pQE9. This construct puts a His tag on
the N-terminus
of the predicted mature extracellular domain of D-SLAM. The His tag will be
followed by
the Gly-Ser of the BamHI site, and this will then be followed by A23 of SEQ ID
N0:2. This
construct will continue through D233 of SEQ ID N0:2, and will be followed by a
TAA stop
codon. The amino acid sequence encoded by this construct is as follows:
MRGSHHHHHHGSAQVLSKVGGSVLLVAARPPGFQVREAIWRSLWPSEELLATFFRG
SLETLYHSRFLGRAQLHSNLSLELGPLESGDSGNFS VLMVDTRGQPWTQTLQLKV YD
AVPRPV VQVFIAVERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLF
TDGQVLSISLGPGDRDVAYSCIVSNPVSWDLATVTPWDSCHHEAAPGKASYKD
(SEQ ID NO: 19).
Additionally, a mature form containing only the extracellular domain of D-SLAM
(amino acids A23 to D233 of SEQ ID N0:2) can also be inserted into an E. coli
expression
vector, such as pHE4 (see below). In this example, the 5' primer, containing a
Nde
restriction site, indicated in bold, can be used:
GCAGCACATATGGCCCAAGTGCTGAGCAAAGTCG (SEQ ID NO: 20) and a 3' primer
containing an Asp718 restriction site, shown in bold, can be used:
GCAGCAGGTACCTTACTAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ
ID NO: 21).
Clones containing the desired constructs are grown overnight (0/N) in liquid
culture
in LB media supplemented with both Amp ( 100 ug/ml) and Kan (25 ug/ml). The
O/N culture
is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells
are grown to an
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optical density 600 (O.D.~"") of between 0.4 and 0.6. IPTG (Isopropyl-B-D-
thiogalacto
pyranoside) is then added to a final concentration of 1 mM. IPTG induces by
inactivating the
lacI repressor, clearing the P/O leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by
centrifugation
(20 mins at 6000Xg). The cell pellet is solubilized in the chaotropic agent 6
Molar Guanidine
HCl by stirring for 3-4 hours at 4~C. The cell debris is removed by
centrifugation, and the
supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-
acetic acid ("Ni
NTA") affinity resin column (available from QIAGEN, Inc., supra). Proteins
with a 6 x His
tag bind to the Ni-NTA resin with high affinity and can be purified in a
simple one-step
procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCI, pH 8,
the
column is first washed with 10 volumes of 6 M guanidine-HCI, pH 8, then washed
with 10
volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with
6 M
guanidine-HCI, pH 5.
The purified D-SLAM protein is then renatured by dialyzing it against
phosphate-
buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI.
Alternatively,
the D-SLAM protein can be successfully refolded while immobilized on the Ni-
NTA column.
The recommended conditions are as follows: renature using a linear 6M-1M urea
gradient in
500 mM NaCI, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease
inhibitors. The
renaturation should be performed over a period of 1.5 hours or more. After
renaturation the
proteins are eluted by the addition of 250 mM immidazole. Immidazole is
removed by a final
dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM
NaCI. The
purified D-SLAM protein is stored at 4 ~C or frozen at -80~C.
In addition to the above expression vector, the present invention further
includes an
expression vector comprising phage operator and promoter elements operatively
linked to a
D-SLAM polynucleotide, called pHE4a. (ATCC Accession Number 209645, deposited
February 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene
as a
selection marker, 2) an E. coli origin of replication, 3) a TS phage promoter
sequence, 4) two
lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose
operon repressor
gene (lacIq). The origin of replication (oriC) is derived from pUCl9 (LTI,
Gaithersburg,
MD). The promoter sequence and operator sequences are made synthetically.
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DNA can be inserted into the pHEa by restricting the vector with NdeI and
XbaI,
BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating
the larger
fragment (the stuffer fragment should be about 310 base pairs). The DNA insert
is generated
according to the PCR protocol described in Example l, using PCR primers having
restriction
sites for NdeI (5' primer) and XbaI, BamHI, XhoI, or Asp718 (3' primer). The
PCR insert is
gel purified and restricted with compatible enzymes. The insert and vector are
ligated
according to standard protocols.
The engineered vector could easily be substituted in the above protocol to
express
protein in a bacterial system.
Example 6: Purification of D-SLAM Polypeptide from an Inclusion Body
The following alternative method can be used to purify D-SLAM polypeptide
expressed in E coli when it is present in the form of inclusion bodies. Unless
otherwise
specified, all of the following steps are conducted at 4-10~C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture
is cooled to 4-10~C and the cells harvested by continuous centrifugation at
15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein per unit
weight of cell paste
and the amount of purified protein required, an appropriate amount of cell
paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The
cells are
dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The
homogenate is then
mixed with NaCI solution to a final concentration of 0.5 M NaCI, followed by
centrifugation
at 7000 xg for 15 min. The resultant pellet is washed again using O.SM NaCI,
100 mM Tris,
50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride (GuHCI) for 2-4 hours. After 7000 xg centrifugation for 15 min.,
the pellet is
discarded and the polypeptide containing supernatant is incubated at 4~C
overnight to allow
further GuHCI extraction.
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Following high speed centrifugation (30,000 xg) to remove insoluble particles,
the
GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract with
20 volumes
of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by vigorous
stirring. The refolded diluted protein solution is kept at 4~C without mixing
for 12 hours
prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential
filtration
unit equipped with 0.16 um membrane filter with appropriate surface area
(e.g., Filtron),
equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered
sample is loaded
onto a canon exchange resin (e.g., Poros HS-50, Perceptive Biosystems). The
column is
washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000
mM,
and 1500 mM NaCI in the same buffer, in a stepwise manner. The absorbance at
280 nm of
the effluent is continuously monitored. Fractions are collected and further
analyzed by SDS-
PAGE.
Fractions containing the D-SLAM polypeptide are then pooled and mixed with 4
volumes of water. The diluted sample is then loaded onto a previously prepared
set of
tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak
anion
(Poros CM-20, Perceptive Biosystems) exchange resins. The columns are
equilibrated with
40 mM sodium acetate, pH 6Ø Both columns are washed with 40 mM sodium
acetate, pH
6.0, 200 mM NaCI. The CM-20 column is then eluted using a 10 column volume
linear
gradient ranging from 0.2 M NaCI, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCI,
50 mM
sodium acetate, pH 6.5. Fractions are collected under constant AzR" monitoring
of the
effluent. Fractions containing the polypeptide (determined, for instance, by
16% SDS-
PAGE) are then pooled.
The resultant D-SLAM polypeptide should exhibit greater than 95% purity after
the
above refolding and purification steps. No major contaminant bands should be
observed
from Commassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is
loaded.
The purified D-SLAM protein can also be tested for endotoxin/LPS
contamination, and
typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 7: Cloning and Expression of D-SLAM in a Baculovirus Expression System
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In this example, the plasmid shuttle vector pA2 is used to insert D-SLAM
polynucleotide into a baculovirus to express D-SLAM. This expression vector
contains the
strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis
virus
(AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and
Asp718. The
polyadenylation site of the simian virus 40 ("SV40") is used for efficient
polyadenylation.
For easy selection of recombinant virus, the plasmid contains the beta-
galactosidase gene
from E. coli under control of a weak Drosophila promoter in the same
orientation, followed
by the polyadenylation signal of the polyhedrin gene. The inserted genes are
flanked on both
sides by viral sequences for cell-mediated homologous recombination with wild-
type viral
DNA to generate a viable virus that express the cloned D-SLAM polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such
as
pAc373, pVL941, and pAcIMI, as one skilled in the art would readily
appreciate, as long as
the construct provides appropriately located signals for transcription,
translation, secretion
and the like, including a signal peptide and an in-frame AUG as required. Such
vectors are
described, for instance, in Luckow et al., Virology 170:31-39 ( 1989).
Specifically, the D-SLAM cDNA sequence contained in the deposited clone,
including the AUG initiation codon and any naturally associated leader
sequence, is amplified
using the PCR protocol described in Example 1. If the naturally occurring
signal sequence is
used to produce the secreted protein, the pA2 vector does not need a second
signal peptide.
Alternatively, the vector can be modified (pA2 GP) to include a baculovirus
leader sequence,
using the standard methods described in Summers et al., "A Manual of Methods
for
Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural
Experimental
Station Bulletin No. 1555 (1987).
Fragments of D-SLAM can be expressed from the baculovirus system. For example,
the predicted extracellular domain (M1-K232 of SEQ ID N0:2) can be inserted
into pA2
using the primers described throughout the Example section.
The amplified fragment is isolated from a 1 % agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is
digested with
appropriate restriction enzymes and again purified on a 1 % agarose gel.
The plasmid is digested with the corresponding restriction enzymes and
optionally,
can be dephosphorylated using calf intestinal phosphatase, using routine
procedures known in
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the art. The DNA is then isolated from a 1% agarose gel using a commercially
available kit
("Geneclean" BIO 101 Inc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA
ligase. E. coli HB 101 or other suitable E. coli hosts such as XL-1 Blue
(Stratagene Cloning
Systems, La Jolla, CA) cells are transformed with the ligation mixture and
spread on culture
plates. Bacteria containing the plasmid are identified by digesting DNA from
individual
colonies and analyzing the digestion product by gel electrophoresis. The
sequence of the
cloned fragment is confirmed by DNA sequencing.
Five ug of a plasmid containing the polynucleotide is co-transfected with 1.0
ug of a
commercially available linearized baculovirus DNA ("BaculoGoldTr'' baculovirus
DNA",
Pharmingen, San Diego, CA), using the lipofection method described by Felgner
et al., Proc.
Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldTM virus DNA and
5 ug
of the plasmid are mixed in a sterile well of a microtiter plate containing 50
u1 of serum-free
Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards, 10 u1
Lipofectin
plus 90 u1 Grace's medium are added, mixed and incubated for 15 minutes at
room
temperature. Then the transfection mixture is added drop-wise to Sf9 insect
cells (ATCC
CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium
without serum.
The plate is then incubated for 5 hours at 27 degrees C. The transfection
solution is then
removed from the plate and 1 ml of Grace's insect medium supplemented with 10%
fetal calf
serum is added. Cultivation is then continued at 27 degrees C for four days.
After four days the supernatant is collected and a plaque assay is performed,
as
described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life
Technologies
Inc., Gaithersburg) is used to allow easy identification and isolation of gal-
expressing clones,
which produce blue-stained plaques. (A detailed description of a "plaque
assay" of this type
can also be found in the user's guide for insect cell culture and
baculovirology distributed by
Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate
incubation, blue stained
plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar
containing the
recombinant viruses is then resuspended in a microcentrifuge tube containing
200 u1 of
Grace's medium and the suspension containing the recombinant baculovirus is
used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these
culture dishes are
harvested and then they are stored at 4~C.
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To verify the expression of the polypeptide, Sf9 cells are grown in Grace's
medium
supplemented with 10% heat-inactivated FBS. The cells are infected with the
recombinant
baculovirus containing the polynucleotide at a multiplicity of infection
("MOI") of about 2.
If radiolabeled proteins are desired, 6 hours later the medium is removed and
is replaced with
SF900 II medium minus methionine and cysteine (available from Life
Technologies Inc.,
Rockville, MD). After 42 hours, 5 uCi of ASS-methionine and 5 uCi 35S-cysteine
(available
from Amersham) are added. The cells are further incubated for 16 hours and
then are
harvested by centrifugation. The proteins in the supernatant as well as the
intracellular
proteins are analyzed by SDS-PAGE followed by autoradiography (if
radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified
protein may be used to determine the amino terminal sequence of the produced D-
SLAM
protein.
Example 8: Expression of D-SLAM in Mammalian Cells
D-SLAM polypeptide can be expressed in a mammalian cell. A typical mammalian
expression vector contains a promoter element, which mediates the initiation
of transcription
of mRNA, a protein coding sequence, and signals required for the termination
of transcription
and polyadenylation of the transcript. Additional elements include enhancers,
Kozak
sequences and intervening sequences flanked by donor and acceptor sites for
RNA splicing.
Highly efficient transcription is achieved with the early and late promoters
from SV40, the
long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early
promoter of the cytomegalovirus (CMV). However, cellular elements can also be
used (e.g.,
the human actin promoter).
Suitable expression vectors for use in practicing the present invention
include, for
example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat
(ATCC
37152), pSV2DHFR (ATCC 37146), pBCI2MI (ATCC 67109), pCMVSport 2.0, and
pCMVSport 3Ø Mammalian host cells that could be used include, human Hela,
293, H9 and
Jurkat cells, mouse NIH3T3 and C127 cells, Cos l, Cos 7 and CV1, quail QC1-3
cells, mouse
L cells and Chinese hamster ovary (CHO) cells.
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Alternatively, D-SLAM polypeptide can be expressed in stable cell lines
containing
the D-SLAM polynucleotide integrated into a chromosome. The co-transfection
with a
selectable marker such as DHFR, gpt, neomycin, hygromycin allows the
identification and
isolation of the transfected cells.
The transfected D-SLAM gene can also be amplified to express large amounts of
the
encoded protein. The DHFR (dihydrofolate reductase) marker is useful in
developing cell
lines that carry several hundred or even several thousand copies of the gene
of interest. (See,
e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L.
and Ma, C.,
Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M.
A.,
Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme
glutamine
synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et
al.,
Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are
grown in
selective medium and the cells with the highest resistance are selected. These
cell lines
contain the amplified genes) integrated into a chromosome. Chinese hamster
ovary (CHO)
and NSO cells are often used for the production of proteins.
Derivatives of the plasmid pSV2-DHFR (ATCC Accession No. 37146), the
expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession
No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen
et al.,
Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-
enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites,
e.g., with the
restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the
cloning of D-
SLAM. The vectors also contain the 3' intron, the polyadenylation and
termination signal of
the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40
early
promoter.
Specifically, the plasmid pC6 or pC4 is digested appropriate restriction
enzymes and then
dephosphorylated using calf intestinal phosphates by procedures known in the
art. The vector
is then isolated from a 1 % agarose gel.
D-SLAM polynucleotide is amplified according to the protocol outlined in
Example
1. If a naturally occurring signal sequence is used to produce a secreted
protein, the vector
does not need a second signal peptide. Alternatively, if a naturally occurring
signal sequence
is not used, the vector can be modified to include a heterologous signal
sequence in an effort
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to secrete the protein from the cell. (See, e.g., WO 96/34891.)
Specifically, the full length D-SLAM protein can be expressed from a mammalian
vector, such as pC4, using the following primers: The 5' primer, containing a
BamHI in bold,
is as follows:
S GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC
(SEQ ID N0:22) while the 3' primer contains a Xba site shown in bold:
GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO:
23). This construct should produce a transmembrane protein that will be
expressed on the
external cell surface.
Alternatively, a construct containing only the soluble portion of D-SLAM can
be
made by inserting the predicted extracellular domain of D-SLAM in pC4. For
example,
DNA encoding M1-K232 of SEQ ID N0:2 can be in inserted into pC4, using a 5'
primer,
containing a BamHI restriction site shown in bold:
GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ
ID NO: 22) and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:
24).
The amplified fragment is isolated from a 1 % agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is
digested with
appropriate restriction enzymes and again purified on a 1 % agarose gel.
The amplified fragment is then digested with the same restriction enzyme and
purified on a
1 % agarose gel. The isolated fragment and the dephosphorylated vector are
then ligated with
T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and
bacteria are
identified that contain the fragment inserted into plasmid pC6 or pC4 using,
for instance,
restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for
transfection.
Five pg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 ug of
the plasmid
pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains
a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that confers
resistance to a
group of antibiotics including 6418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml 6418. After 2 days, the cells are trypsinized and
seeded in
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hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with 10,
25, or 50 ng/ml of metothrexate plus 1 mg/ml 6418. After about 10-14 days
single clones
are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using
different
concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing
at the highest concentrations of methotrexate are then transferred to new 6-
well plates
containing even higher concentrations of methotrexate ( 1 uM, 2 uM, 5 uM, 10
mM, 20 mM).
The same procedure is repeated until clones are obtained which grow at a
concentration of
100 - 200 uM. Expression of D-SLAM is analyzed, for instance, by SDS-PAGE and
Western
blot or by reversed phase HPLC analysis.
Example 9: Construction of N Terminal and/or C-Terminal Deletion Mutants
The following general approach may be used to clone a N-terminal or C-terminal
deletion D-SLAM deletion mutant. Generally, two oligonucleotide primers of
about 15-25
nucleotides are derived from the desired 5' and 3' positions of a
polynucleotide of SEQ ID
NO:1. The 5' and 3' positions of the primers are determined based on the
desired D-SLAM
polynucleotide fragment. An initiation and stop codon are added to the 5' and
3' primers
respectively, if necessary, to express the D-SLAM polypeptide fragment encoded
by the
polynucleotide fragment. Preferred D-SLAM polynucleotide fragments are those
encoding
the N-terminal and C-terminal deletion mutants disclosed above in the
"Polynucleotide and
Polypeptide Fragments" section of the Specification.
Additional nucleotides containing restriction sites to facilitate cloning of
the D-SLAM
polynucleotide fragment in a desired vector may also be added to the 5' and 3'
primer
sequences. The D-SLAM polynucleotide fragment is amplified from genomic DNA or
from
the deposited cDNA clone using the appropriate PCR oligonucleotide primers and
conditions
discussed herein or known in the art. The D-SLAM polypeptide fragments encoded
by the
D-SLAM polynucleotide fragments of the present invention may be expressed and
purified in
the same general manner as the full length polypeptides, although routine
modifications may
be necessary due to the differences in chemical and physical properties
between a particular
fragment and full length polypeptide.
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As a means of exemplifying but not limiting the present invention, the
polynucleotide
encoding the D-SLAM polypeptide fragment Leu-35 to Thr-276 is amplified and
cloned as
follows: A 5' primer is generated comprising a restriction enzyme site
followed by an
initiation codon in frame with the polynucleotide sequence encoding the N-
terminal portion
of the polypeptide fragment beginning with Leu-35. A complementary 3' primer
is generated
comprising a restriction enzyme site followed by a stop codon in frame with
the
polynucleotide sequence encoding C-terminal portion of the D-SLAM polypeptide
fragment
ending with Thr-276.
The amplified polynucleotide fragment and the expression vector are digested
with
restriction enzymes which recognize the sites in the primers. The digested
polynucleotides
are then ligated together. The D-SLAM polynucleotide fragment is inserted into
the
restricted expression vector, preferably in a manner which places the D-SLAM
polypeptide
fragment coding region downstream from the promoter. The ligation mixture is
transformed
into competent E. coli cells using standard procedures and as described in the
Examples
herein. Plasmid DNA is isolated from resistant colonies and the identity of
the cloned DNA
confirmed by restriction analysis, PCR and DNA sequencing.
Example 10: Protein Fusions of D-SLAM
D-SLAM polypeptides are preferably fused to other proteins. These fusion
proteins
can be used for a variety of applications. For example, fusion of D-SLAM
polypeptides to
His-tag, HA-tag, protein A, IgG domains, and maltose binding protein
facilitates purification.
(See Example 5; see also EP A 394,827; Traunecker, et al., Nature 331:84-86
(1988).)
Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in
vivo. Nuclear
localization signals fused to D-SLAM polypeptides can target the protein to a
specific
subcellular localization, while covalent heterodimer or homodimers can
increase or decrease
the activity of a fusion protein. Fusion proteins can also create chimeric
molecules having
more than one function. Finally, fusion proteins can increase solubility
and/or stability of the
fused protein compared to the non-fused protein. All of the types of fusion
proteins described
above can be made by modifying the following protocol, which outlines the
fusion of a
polypeptide to an IgG molecule, or the protocol described in Example 5.
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Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using
primers that span the 5' and 3' ends of the sequence described below. These
primers also
should have convenient restriction enzyme sites that will facilitate cloning
into an expression
vector, preferably a mammalian expression vector.
For example, if pC4 (Accession No. 209646) is used, the human Fc portion can
be
ligated into the BamHI cloning site. Note that the 3' BamHI site should be
destroyed. Next,
the vector containing the human Fc portion is re-restricted with BamHI,
linearizing the
vector, and D-SLAM polynucleotide, isolated by the PCR protocol described in
Example 1, is
ligated into this BamHI site. Note that the polynucleotide is cloned without a
stop codon,
otherwise a fusion protein will not be produced.
Examples of primers that can be used to amplify D-SLAM polypeptides, such as
the
predicted extracellular domain of D-SLAM include: a 5' primer containing a
BamHI
restriction site shown in bold:
GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ
ID NO: 22) and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:
25). Using these primers, the construct will express the predicted
extracellular domain of D-
SLAM fused to Fc in pC4.
If the naturally occurring signal sequence is used to produce the secreted
protein, pC4
does not need a second signal peptide. Alternatively, if the naturally
occurring signal
sequence is not used, the vector can be modified to include a heterologous
signal sequence.
(See, e.g., WO 96/34891.)
Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAG
CACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA
CACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC
AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCC
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AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT
GAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID N0:4)
Alternatively, this same region can be inserted in pA2, creating a fusion of
Fc with the
predicted extracellular domain of D-SLAM. Examples of primer that can be used
to amplify
the predicted extracellular domain of D-SLAM include: a 5' primer containing a
BamHI
restriction site shown in bold:
GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC(SEQ
ID NO: 22) and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:
25). This construct will express the predicted extracellular domain of D-SLAM
fused to Fc
in pA2.
These two above constructs were expressed in their appropriate host cells
(e.g., pC4 in
a mammalian system, while pA2 in a baculovirus system), both systems produced
a truncated
protein, lacking the predicted signal sequence and beginning with A23 of SEQ
ID N0:2.
Thus, as was predicted, both baculovirus and mammalian systems process D-SLAM
to amino
acid A23 of SEQ ID N0:2. However, due to differences in glycosylation, the
baculovirus
system produces a protein, under reducing conditions, migrating at around 60
kD, while the
mammalian system generates a protein migrating at around 55 kD.
Additionally, fusion proteins of D-SLAM polypeptides, including the predicted
extracellular domain or the mature form of D-SLAM, can be fused to FLAG for
mammalian
cell expression. For example, the extracellular domain can be amplifed using
the following
primers: a 5' primer containing a BamHI restriction site shown in bold:
GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC(SEQ
ID NO: 22) and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGATTACTTGTCATCGTCGTCCTTGTAGTCATCTTTGTAGGAGGCC
TTCCCTGGTGCTG (SEQ ID NO: 26).
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Example Il: Production of an Antibody
a) Hybridoma Technology
The antibodies of the present invention can be prepared by a variety of
methods.
(See, Current Protocols, Chapter 2.) As one example of such methods, cells
expressing D-
SLAM is administered to an animal to induce the production of sera containing
polyclonal
antibodies. In a preferred method, a preparation of D-SLAM protein is prepared
and purified
to render it substantially free of natural contaminants. Such a preparation is
then introduced
into an animal in order to produce polyclonal antisera of greater specific
activity.
In the most preferred method, the antibodies of the present invention are
monoclonal
antibodies (or protein binding fragments thereof). Such monoclonal antibodies
can be
prepared using hybridoma technology. (Kohler et al., Nature 256:495 ( 1975);
Kohler et al.,
Eur. J. Immunol. 6:511 ( 1976); Kohler et al., Eur. J. Immunol. 6:292 ( 1976);
Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-
681 ( 1981 ).)
In general, such procedures involve immunizing an animal (preferably a mouse)
with D-
SLAM polypeptide or, more preferably, with a secreted D-SLAM polypeptide-
expressing
cell. Such cells may be cultured in any suitable tissue culture medium;
however, it is
preferable to culture cells in Earle's modified Eagle's medium supplemented
with 10% fetal
bovine serum (inactivated at about 56~C), and supplemented with about 10 g/1
of nonessential
amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of
streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell
line. Any suitable myeloma cell line may be employed in accordance with the
present
invention; however, it is preferable to employ the parent myeloma cell line
(SP20), available
from the ATCC. After fusion, the resulting hybridoma cells are selectively
maintained in
HAT medium, and then cloned by limiting dilution as described by Wands et al.
(Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through
such a
selection are then assayed to identify clones which secrete antibodies capable
of binding the
D-SLAM polypeptide.
Alternatively, additional antibodies capable of binding to D-SLAM polypeptide
can
be produced in a two-step procedure using anti-idiotypic antibodies. Such a
method makes
use of the fact that antibodies are themselves antigens, and therefore, it is
possible to obtain
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an antibody which binds to a second antibody. In accordance with this method,
protein
specific antibodies are used to immunize an animal, preferably a mouse. The
splenocytes of
such an animal are then used to produce hybridoma cells, and the hybridoma
cells are
screened to identify clones which produce an antibody whose ability to bind to
the D-SLAM
protein-specific antibody can be blocked byD-SLAM. Such antibodies comprise
anti-
idiotypic antibodies to the D-SLAM protein-specific antibody and can be used
to immunize
an animal to induce formation of further D-SLAM protein-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the
antibodies of the
present invention may be used according to the methods disclosed herein. Such
fragments
are typically produced by proteolytic cleavage, using enzymes such as papain
(to produce Fab
fragments) or pepsin (to produce F(ab')2 fragments). Alternatively, secreted D-
SLAM
protein-binding fragments can be produced through the application of
recombinant DNA
technology or through synthetic chemistry.
For in vivo use of antibodies in humans, it may be preferable to use
"humanized"
chimeric monoclonal antibodies. Such antibodies can be produced using genetic
constructs
derived from hybridoma cells producing the monoclonal antibodies described
above.
Methods for producing chimeric antibodies are known in the art. (See, for
review, 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).)
b) Isolation of antibody fragments directed against D-SLAM from a library of
scFvs.
Naturally occuring V-genes isolated from human PBLs are constructed into a
large
library of antibody fragments which contain reactivities against D-SLAM to
which the donor
may or may not have been exposed (see e.g., U.S. Patent 5,885,793 incorporated
herein in its
entirety by reference).
Rescue of the Library. A library of scFvs is constructed from the RNA of human
PBLs as described in W092/01047. To rescue phage displaying antibody
fragments,
approximately 10y E. coli harbouring the phagemid are used to inoculate 50 ml
of 2xTY
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containing 1 % glucose and 100 ug/ml of ampicillin (2xTY-AMP-GLU) and grown to
an O.D.
of 0.8 with shaking. Five ml of this culture is used to innoculate 50 ml of
2xTY-AMP-GLU,
2 x lOR TU of delta gene 3 helper (M13 delta gene III, see W092/01047) are
added and the
culture incubated at 37~C for 45 minutes without shaking and then at 37~C for
45 minutes
with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the
pellet resuspended
in 2 liters of of 2xTY containing 100 ug/ml ampicillin and 50 ug/ml kanamycin
and grown
overnight. Phage are prepared as described in W092/01047.
M 13 delta gene III is prepared as follows: M 13 delta gene III helper phage
does not
encode gene III protein, hence the phage(mid) displaying antibody fragments
have a greater
avidity of binding to antigen. Infectious M 13 delta gene III particles are
made by growing the
helper phage in cells harbouring a pUC 19 derivative supplying the wild type
gene III protein
during phage morphogenesis. The culture is incubated for 1 hour at 37~C
without shaking and
then for a further hour at 37~C with shaking. Cells are spun down (IEC-Centra
8, 4000
revs/min for 10 min), resuspended in 300 ml 2xTY broth containing 100 ug
ampicillin/ml and
25 ug kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37°
C. Phage
particles are purified and concentrated from the culture medium by two PEG-
precipitations
(Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 um
filter
(Minisart NML; Sartorius) to give a final concentration of approximately 10"
transducing
units/ml (ampicillin-resistant clones).
Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4
ml
of either 100 ug/ml or 10 ug/ml of a polypeptide of the present invention.
Tubes are blocked
with 2% Marvel-PBS for 2 hours at 37~C and then washed 3 times in PBS.
Approximately
10'3 TU of phage is applied to the tube and incubated for 30 minutes at room
temperature
tumbling on an over and under turntable and then left to stand for another 1.5
hours. Tubes
are washed 10 times with PBS 0.1 % Tween-20 and 10 times with PBS. Phage are
eluted by
adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and
over turntable
after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-
HCI, pH 7.4.
Phage are then used to infect 10 ml of mid-log E. coli TG 1 by incubating
eluted phage with
bacteria for 30 minutes at 37~C. The E. coli are then plated on TYE plates
containing 1%
glucose and 100 ug/ml ampicillin. The resulting bacterial library is then
rescued with delta
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gene 3 helper phage as described above to prepare phage for a subsequent round
of selection.
This process is then repeated for a total of 4 rounds of affinity purification
with tube-washing
increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds
3 and 4.
S Characterization of Binders. Eluted phage from the 3rd and 4th rounds of
selection
are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et
al., 1991) from
single colonies for assay. ELISAs are performed with microtitre plates coated
with either 10
pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6.
Clones
positive in ELISA are further characterized by PCR fingerprinting (see e.g.,
W092/01047)
and then by sequencing.
Example 12: Production Of D-SLAM Protein For High-Throughput Screening Assays
The following protocol produces a supernatant containing D-SLAM polypeptide to
be
tested. This supernatant can then be used in the Screening Assays described in
Examples 14
21.
First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution
(lmg/ml
in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a
working
solution of SOug/ml. Add 200 u1 of this solution to each well (24 well plates)
and incubate at
RT for 20 minutes. Be sure to distribute the solution over each well (note: a
12-channel
pipetter may be used with tips on every other channel). Aspirate off the Poly-
D-Lysine
solution and rinse with lml PBS (Phosphate Buffered Saline). The PBS should
remain in the
well until just prior to plating the cells and plates may be poly-lysine
coated in advance for up
to two weeks.
Plate 293T cells (do not carry cells past P+20) at 2 x 105 cells/well in .5m1
DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine
(12-
604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/lx
Penstrep(17-602E
Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 u1 Lipofectamine (
18324
012 Gibco/BRL) and 5m1 Optimem I (31985070 GibcoBRL)/96-well plate. With a
small
volume multi-channel pipetter, aliquot approximately tug of an expression
vector containing
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a polynucleotide insert, produced by the methods described in Examples 8-10,
into an
appropriately labeled 96-well round bottom plate. With a mufti-channel
pipetter, add 50u1 of
the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently
to mix.
Incubate at RT 15-45 minutes. After about 20 minutes, use a mufti-channel
pipetter to add
150u1 Optimem I to each well. As a control, one plate of vector DNA lacking an
insert
should be transfected with each set of transfections.
Preferably, the transfection should be performed by tag-teaming the following
tasks.
By tag-teaming, hands on time is cut in half, and the cells do not spend too
much time on
PBS. First, person A aspirates off the media from four 24-well plates of
cells, and then
person B rinses each well with .5-lml PBS. Person A then aspirates off PBS
rinse, and
person B, using a12-channel pipetter with tips on every other channel, adds
the 200u1 of
DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even
wells, to
each row on the 24-well plates. Incubate at 37~C for 6 hours.
While cells are incubating, prepare appropriate media, either 1%BSA in DMEM
with
1 x penstrep, or HGS CHO-5 media ( 116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L
CuS04-
5H20; 0.050 mg/L of Fe(N03)3-9H20; 0.417 mg/L of FeS04-7H20; 311.80 mg/L of
Kcl;
28.64 mg/L of MgCl2; 48.84 mg/L of MgS04; 6995.50 mg/L of NaCI; 2400.0 mg/L of
NaHC03; 62.50 mg/L of NaH2P04-H20; 71.02 mg/L of Na2HP04; .4320 mg/L of ZnS04-
7H20; .002 mg/L of Arachidonic Acid ; 1.022 mg/L of Cholesterol; .070 mg/L of
DL-alpha-
Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic
Acid; 0.010
mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid;
0.010 mg/L
of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20
mg/L of
Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of
L-
Arginine-HCL; 7.50 mg/ml of L-Asparagine-H20; 6.65 mg/ml of L-Aspartic Acid;
29.56
mg/ml of L-Cystine-2HCL-H20; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-
Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml
of L-
Histidine-HCL-H20; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine;
163.75
mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-
Phenylalainine;
40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H20; and 99.65 mg/ml of
L-
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Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of
Choline
Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of
Niacinamide; 3.00
mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;
3.17
mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin B 12; 25
mM of
HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081
mg/L of
Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium
Selenite;
20uM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-
Cyclodextrin
complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed
with Oleic
Acid; 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal Acetate. Adjust
osmolarity to 327 mOsm) with 2mm glutamine and lx penstrep. (BSA (81-068-3
Bayer)
100gm dissolved in 1L DMEM for a 10% BSA stock solution). Filter the media and
collect
50 u1 for endotoxin assay in 15m1 polystyrene conical.
The transfection reaction is terminated, preferably by tag-teaming, at the end
of the
incubation period. Person A aspirates off the transfection media, while person
B adds 1.5m1
appropriate media to each well. Incubate at 37~C for 45 or 72 hours depending
on the media
used: 1 %BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300u1 multichannel pipetter, aliquot 600u1 in one lml
deep well
plate and the remaining supernatant into a 2m1 deep well. The supernatants
from each well
can then be used in the assays described in Examples 14-21.
It is specifically understood that when activity is obtained in any of the
assays
described below using a supernatant, the activity originates from either the D-
SLAM
polypeptide directly (e.g., as a secreted protein) or by D-SLAM inducing
expression of other
proteins, which are then secreted into the supernatant. Thus, the invention
further provides a
method of identifying the protein in the supernatant characterized by an
activity in a
particular assay.
Example 13: Construction of GAS Reporter Construct
One signal transduction pathway involved in the differentiation and
proliferation of
cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs
pathway bind
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to gamma activation site "GAS" elements or interferon-sensitive responsive
element
("ISRE"), located in the promoter of many genes. The binding of a protein to
these elements
alter the expression of the associated gene.
GAS and ISRE elements are recognized by a class of transcription factors
called
Signal Transducers and Activators of Transcription, or "STATs." There are six
members of
the STATs family. Statl and Stat3 are present in many cell types, as is Stat2
(as response to
IFN-alpha is widespread). Stat4 is more restricted and is not in many cell
types though it has
been found in T helper class I, cells after treatment with IL-12. StatS was
originally called
mammary growth factor, but has been found at higher concentrations in other
cells including
myeloid cells. It can be activated in tissue culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus upon
tyrosine phosphorylation by a set of kinases known as the Janus Kinase
("Jaks") family. Jaks
represent a distinct family of soluble tyrosine kinases and include Tyk2,
Jakl, Jak2, and Jak3.
These kinases display significant sequence similarity and are generally
catalytically inactive
in resting cells.
The Jaks are activated by a wide range of receptors summarized in the Table
below.
(Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51
(1995).) A
cytokine receptor family, capable of activating Jaks, is divided into two
groups: (a) Class 1
includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-
15, Epo, PRL, GH,
G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a,
IFN-g,
and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of
four conserved
cysteines and one tryptophan) and a WSXWS motif (a membrane proxial region
encoding
Trp-Ser-Xxx-Ttp-Ser (SEQ ID NO:S)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn
activate
STATs, which then translocate and bind to GAS elements. This entire process is
encompassed in the Jaks-STATs signal transduction pathway.
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of
the GAS
or the ISRE element, can be used to indicate proteins involved in the
proliferation and
differentiation of cells. For example, growth factors and cytokines are known
to activate the
Jaks-STATs pathway. (See Table below.) Thus, by using GAS elements linked to
reporter
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molecules, activators of the Jaks-STATs pathway can be identified.
JAKs STATS GAS(elements)
or ISRE
Ligand tvk2 Jakl Jak2 Jak3
IFN family
IFN-a/B + + - - 1,2,3 ISRE
IFN-g + + - 1 GAS (IRF1>Lys6>IFP)
Il-10 + ? ? - 1,3
gp130 family
IL-6 (Pleiotrohic)+ + ? 1,3 GAS
+ (IRF1>Lys6>IFP)
Il-11(Pleiotrohic)+ ? ? 1,3
?
OnM(Pleiotrohic)+ + ? 1,3
?
LIF(Pleiotrohic)+ + ? 1,3
?
CNTF(Pleiotrohic)+ + ? 1,3
-/+
G-CSF(Pleiotrohic)?+ ? ? 1,3
IL-12(Pleiotrohic)- + + 1,3
+
g-C family
IL-2 (lymphocytes)-+ - + 1,3,5 GAS
2$ IL-4 (lymph/myeloid)- + - + 6 GAS (IRF1 =1FP
Ly6)(IgH)
IL-7 (lymphocytes)-+ - + 5 GAS
IL-9 (lymphocytes)-+ - + 5 GAS
IL-13 (lymphocyte)-+ ? ? 6 GAS
IL-15 ? + ? + 5 GAS
gp140 family
IL-3 (myeloid) - - + - 5 GAS (IRF1>IFPLy6)
IL-5 (myeloid) - - + - 5 GAS
GM-CSF (myeloid)-- + - 5 GAS
Growth hormone
family
GH ? - + - 5
PRL ? +/- + - 1,3,5
EPO ? - + - 5 GAS(B-CAS>IRF1=IFPLy6)
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Receptor Tyrosine Kinases
EGF ? + + - 1,3 GAS (IRF1)
PDGF ? + + - 1,3
CSF-1 ? + + - 1,3 GAS (not IRF1)
To construct a synthetic GAS containing promoter element, which is used in the
Biological Assays described in Examples 14-15, a PCR based strategy is
employed to
generate a GAS-SV40 promoter sequence. The 5' primer contains four tandem
copies of the
GAS binding site found in the IRF1 promoter and previously demonstrated to
bind STATs
upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468
(1994).),
although other GAS or ISRE elements can be used instead. The 5' primer also
contains l8bp
of sequence complementary to the S V40 early promoter sequence and is flanked
with an
XhoI site. The sequence of the 5' primer is:
5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAA
TGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID N0:6)
The downstream primer is complementary to the SV40 promoter and is flanked
with a
Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID N0:7)
PCR amplification is performed using the SV40 promoter template present in the
B-
gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is
digested with
XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward
and
reverse primers confirms that the insert contains the following sequence:
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATT
TCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACT
CCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCT
GACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTC
CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3'
(SEQ ID N0:8)
With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2
reporter construct is next engineered. Here, the reporter molecule is a
secreted alkaline
phosphatase, or "SEAP." Clearly, however, any reporter molecule can be instead
of SEAP,
in this or in any of the other Examples. Well known reporter molecules that
can be used
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instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase,
alkaline
phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein
detectable by
an antibody.
The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned
into the pSEAP-Promoter vector obtained from Clontech using HindIII and XhoI,
effectively
replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to
create the
GAS-SEAP vector. However, this vector does not contain a neomycin resistance
gene, and
therefore, is not preferred for mammalian expression systems.
Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP
reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SaII
and
NotI, and inserted into a backbone vector containing the neomycin resistance
gene, such as
pGFP-1 (Clontech), using these restriction sites in the multiple cloning site,
to create the
GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells,
this vector
can then be used as a reporter molecule for GAS binding as described in
Examples 14-15.
Other constructs can be made using the above description and replacing GAS
with a
different promoter sequence. For example, construction of reporter molecules
containing
NFK-B and EGR promoter sequences are described in Examples 16 and 17. However,
many
other promoters can be substituted using the protocols described in these
Examples. For
instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone
or in
combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS).
Similarly, other cell lines can be used to test reporter construct activity,
such as HELA
(epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC
(aortic), or
Cardiomyocyte.
Example 14: High-Throughput Screening Assay for T cell Activity
The following protocol is used to assess T-cell activity of D-SLAM by
determining
whether D-SLAM supernatant proliferates and/or differentiates T-cells. T-cell
activity is
assessed using the GAS/SEAP/Neo construct produced in Example 13. Thus,
factors that
increase SEAP activity indicate the ability to activate the Jaks-STATS signal
transduction
pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No.
TIB-152),
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although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC
Accession
No. CRL-1582) cells can also be used.
Jurkat T-cells are lymphoblastic CD4+ Thl helper cells. In order to generate
stable
cell lines, approximately 2 million Jurkat cells are transfected with the GAS-
SEAP/neo
vector using DMRIE-C (Life Technologies)(transfection procedure described
below). The
transfected cells are seeded to a density of approximately 20,000 cells per
well and
transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are
expanded and then
tested for their response to increasing concentrations of interferon gamma.
The dose response
of a selected clone is demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 wells
containing
200 u1 of cells. Thus, it is either scaled up, or performed in multiple to
generate sufficient
cells for multiple 96 well plates. Jurkat cells are maintained in RPMI + 10%
serum with
1 %Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of
plasmid
DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 u1 of DMRIE-C and
incubate at
room temperature for 15-45 mins.
During the incubation period, count cell concentration, spin down the required
number of cells ( 10' per transfection), and resuspend in OPTI-MEM to a final
concentration
of 10' cells/ml. Then add lml of 1 x 10' cells in OPTI-MEM to T25 flask and
incubate at
37~C for 6 hrs. After the incubation, add 10 ml of RPMI + 15% serum.
The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI + 10% serum,
1
mg/ml Genticin, and 1 % Pen-Strep. These cells are treated with supernatants
containing D-
SLAM polypeptides or D-SLAM induced polypeptides as produced by the protocol
described
in Example 12.
On the day of treatment with the supernatant, the cells should be washed and
resuspended in fresh RPMI + 10% serum to a density of 500,000 cells per ml.
The exact
number of cells required will depend on the number of supernatants being
screened. For one
96 well plate, approximately 10 million cells (for 10 plates, 100 million
cells) are required.
Transfer the cells to a triangular reservoir boat, in order to dispense the
cells into a 96
well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer
200 u1 of cells into
each well (therefore adding 100, 000 cells per well).
After all the plates have been seeded, 50 u1 of the supernatants are
transferred directly
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from the 96 well plate containing the supernatants into each well using a 12
channel pipette.
In addition, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added
to wells H9,
H 10, and H 11 to serve as additional positive controls for the assay.
The 96 well dishes containing Jurkat cells treated with supernatants are
placed in an
incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 u1
samples from each
well are then transferred to an opaque 96 well plate using a 12 channel
pipette. The opaque
plates should be covered (using sellophene covers) and stored at -20~C until
SEAP assays are
performed according to Example 18. The plates containing the remaining treated
cells are
placed at 4~C and serve as a source of material for repeating the assay on a
specific well if
desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is known
to
activate Jurkat T cells. Over 30 fold induction is typically observed in the
positive control
wells.
Example I5: High-Throughput Screening Assay: Identifying Myeloid Activity
The following protocol is used to assess myeloid activity of D-SLAM by
determining
whether D-SLAM proliferates and/or differentiates myeloid cells. Myeloid cell
activity is
assessed using the GAS/SEAP/Neo construct produced in Example 13. Thus,
factors that
increase SEAP activity indicate the ability to activate the Jaks-STATS signal
transduction
pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell
line, although TF-
1, HL60, or KG 1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced
in
Example 13, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth &
Differentiation, 5:259-265) is used. First, harvest 2x10e7 U937 cells and wash
with PBS.
The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-
inactivated
fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100
mg/ml
streptomycin.
Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing
0.5
mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCI, 5 mM KCI, 375 uM
Na2HP04.7H20, 1 mM MgCl2, and 675 uM CaCl2. Incubate at 37~C for 45 min.
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Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in
ml complete medium and incubate at 37~C for 36 hr.
The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml
6418. The 6418-free medium is used for routine growth but every one to two
months, the
5 cells should be re-grown in 400 ug/ml 6418 for couple of passages.
These cells are tested by harvesting 1x108 cells (this is enough for ten 96-
well plates
assay) and wash with PBS. Suspend the cells in 200 ml above described growth
medium,
with a final density of 5x105 cells/ml. Plate 200 u1 cells per well in the 96-
well plate (or
1x105 cells/well).
10 Add 50 u1 of the supernatant prepared by the protocol described in Example
12.
Incubate at 37 degee C for 48 to 72 hr. As a positive control, 100 Unit/ml
interferon gamma
can be used which is known to activate U937 cells. Over 30 fold induction is
typically
observed in the positive control wells. SEAP assay the supernatant according
to the protocol
described in Example 18.
Example 16: High-Throughput Screening Assay: Identifying Neuronal Activity.
When cells undergo differentiation and proliferation, a group of genes are
activated
through many different signal transduction pathways. One of these genes, EGR1
(early
growth response gene 1 ), is induced in various tissues and cell types upon
activation. The
promoter of EGRI is responsible for such induction. Using the EGR1 promoter
linked to
reporter molecules, activation of cells can be assessed by D-SLAM.
Particularly, the following protocol is used to assess neuronal activity in
PC12 cell
lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate
and/or differentiate
by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol
acetate), NGF
(nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene
expression is
activated during this treatment. Thus, by stably transfecting PC 12 cells with
a construct
containing an EGR promoter linked to SEAP reporter, activation of PC12 cells
by D-SLAM
can be assessed.
The EGR/SEAP reporter construct can be assembled by the following protocol.
The
EGR-1 promoter sequence (-633 to +1)(Sakamoto K et al., Oncogene 6:867-871
(1991)) can
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be PCR amplified from human genomic DNA using the following primers:
5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ ID N0:9)
5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID NO:10)
Using the GAS:SEAP/Neo vector produced in Example 13, EGR1 amplified product
can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using
restriction
enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1
amplified
product with these same enzymes. Ligate the vector and the EGR 1 promoter.
To prepare 96 well-plates for cell culture, two mls of a coating solution (
1:30 dilution
of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter
sterilized)) is
added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed
to air dry for 2
hr.
PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing
10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-inactivated fetal
bovine
serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml
streptomycin on a
precoated 10 cm tissue culture dish. One to four split is done every three to
four days. Cells
are removed from the plates by scraping and resuspended with pipetting up and
down for
more than 15 times.
Transfect the EGR/SEAP/Neo construct into PC 12 using the Lipofectamine
protocol
described in Example 12. EGR-SEAP/PC 12 stable cells are obtained by growing
the cells in
300 ug/ml 6418. The 6418-free medium is used for routine growth but every one
to two
months, the cells should be re-grown in 300 ug/ml 6418 for couple of passages.
To assay for neuronal activity, a 10 cm plate with cells around 70 to 80%
confluent is
screened by removing the old medium. Wash the cells once with PBS (Phosphate
buffered
saline). Then starve the cells in low serum medium (RPMI-1640 containing 1%
horse serum
and 0.5% FBS with antibiotics) overnight.
The next morning, remove the medium and wash the cells with PBS. Scrape off
the
cells from the plate, suspend the cells well in 2 ml low serum medium. Count
the cell
number and add more low serum medium to reach final cell density as Sx 105
cells/ml.
Add 200 u1 of the cell suspension to each well of 96-well plate (equivalent to
1x105
cells/well). Add 50 u1 supernatant produced by Example 12, 37~C for 48 to 72
hr. As a
positive control, a growth factor known to activate PC 12 cells through EGR
can be used,
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such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of
SEAP is
typically seen in the positive control wells. SEAP assay the supernatant
according to
Example 18.
Example 17: High-Throughput Screening Assay for T cell Activity
NF-KB (Nuclear Factor-kappaB) is a transcription factor activated by a wide
variety
of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40,
lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by
expression of certain viral gene products. As a transcription factor, NF-
kappaB regulates the
expression of genes involved in immune cell activation, control of apoptosis
(NF-kappaB
appears to shield cells from apoptosis), B and T-cell development, anti-viral
and
antimicrobial responses, and multiple stress responses.
In non-stimulated conditions, NF-kappaB is retained in the cytoplasm with I-
kappaB
(Inhibitor kappaB). However, upon stimulation, I-kappaB is phosphorylated and
degraded,
causing NF-kappaB to shuttle to the nucleus, thereby activating transcription
of target genes.
Target genes activated by NF-kappaB include IL-2, IL-6, GM-CSF, ICAM-1 and
class 1
MHC.
Due to its central role and ability to respond to a range of stimuli, reporter
constructs
utilizing the NF-kappaB promoter element are used to screen the supernatants
produced in
Example 12. Activators or inhibitors of NF-kappaB would be useful in treating,
diagnosing,
detecting, and/or preventing diseases. For example, inhibitors of NF-kappaB
could be used
to treat, diagnose, detect, and/or prevent those diseases related to the acute
or chronic
activation of NF-kappaB, such as rheumatoid arthritis.
To construct a vector containing the NF-kappaB promoter element, a PCR based
strategy is employed. The upstream primer contains four tandem copies of the
NF-kappaB
binding site (GGGGACTTTCCC) (SEQ ID NO:11), 18 by of sequence complementary to
the
5' end of the SV40 early promoter sequence, and is flanked with an XhoI site:
5'-GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC
TTTCCATCCTGCCATCTCAATTA G-3' (SEQ ID N0:12)
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The downstream primer is complementary to the 3' end of the SV40 promoter and
is
flanked with a Hind III site: 5'-GCG GCA AGC TTT TTG CAA AGC CTA GGC-3' (SEQ
ID N0:7).
PCR amplification is performed using the SV40 promoter template present in the
pB-
gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is
digested with
XhoI and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the
T7 and T3
primers confirms the insert contains the following sequence:
5'-CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC
ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCC
CGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTT
TTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT
GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT-3' (SEQ ID N0:13).
Next, replace the S V40 minimal promoter element present in the pSEAP2-
promoter
plasmid (Clontech) with this NF-kappaB/SV40 fragment using XhoI and HindIII.
However,
this vector does not contain a neomycin resistance gene, and therefore, is not
preferred for
mammalian expression systems.
In order to generate stable mammalian cell lines, the NF-kappaB/SV40/SEAP
cassette
is removed from the above NF-kappaB/SEAP vector using restriction enzymes SaII
and NotI,
and inserted into a vector containing neomycin resistance. Particularly, the
NF-
kappaB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the
GFP gene,
after restricting pGFP-1 with SaII and NotI.
Once NF-kappaB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are
created
and maintained according to the protocol described in Example 14. Similarly,
the method for
assaying supernatants with these stable Jurkat T-cells is also described in
Example 14. As a
positive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H
10, and H 11,
with a 5-10 fold activation typically observed.
Example l8: Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 14-17, SEAP
activity is
assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the
following general
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procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and
Reaction Buffers
used below.
Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 u1 of 2.5x
dilution
buffer into Optiplates containing 35 u1 of a supernatant. Seal the plates with
a plastic sealer
and incubate at 65~C for 30 min. Separate the Optiplates to avoid uneven
heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and
prime
with the Assay Buffer. Add 50 microliters Assay Buffer and incubate at room
temperature 5
min. Empty the dispenser and prime with the Reaction Buffer (see the table
below). Add 50
u1 Reaction Buffer and incubate at room temperature for 20 minutes. Since the
intensity of
the chemiluminescent signal is time dependent, and it takes about 10 minutes
to read 5 plates
on luminometer, one should treat 5 plates at each time and start the second
set 10 minutes
later.
Read the relative light unit in the luminometer. Set H12 as blank, and print
the
results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation:
# of Rxn buffer diluent CSPD
plates (ml) (ml)
10 60 3
11 65 3.25
12 70 3.5
13 75 3.75
14 80 4
15 85 4.25
16 90 4.5
17 95 4.75
18 100 5
19 105 5.25
110 5.5
21 115 5.75
22 120 6
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23 125 6.25
24 130 6.5
25 135 6.75
26 140 7
27 145 7.25
28 150 7.5
29 155 7.75
30 160 8
31 165 8.25
32 170 8.5
33 175 8.75
34 180 9
35 185 9.25
36 190 9.5
37 195 9.75
38 200 10
39 205 10.25
40 210 10.5
41 215 10.75
42 220 11
43 225 11.25
44 230 11.5
45 235 11.75
46 240 12
47 245 12.25
48 250 12.5
49 255 12.75
50 260 13
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Example 19: High-Throughput Screening Assay Identifying Changes in Small
Molecule
Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of
small
molecules, such as calcium, potassium, sodium, and pH, as well as alter
membrane potential.
These alterations can be measured in an assay to identify supernatants which
bind to
receptors of a particular cell. Although the following protocol describes an
assay for calcium,
this protocol can easily be modified to detect changes in potassium, sodium,
pH, membrane
potential, or any other small molecule which is detectable by a fluorescent
probe.
The following assay uses Fluorometric Imaging Plate Reader ("FLIPR") to
measure
changes in fluorescent molecules (Molecular Probes) that bind small molecules.
Clearly, any
fluorescent molecule detecting a small molecule can be used instead of the
calcium
fluorescent molecule, fluo-3, used here.
For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star
black 96-
well plate with clear bottom. The plate is incubated in a CO, incubator for 20
hours. The
adherent cells are washed two times in Biotek washer with 200 u1 of HBSS
(Hank's Balanced
Salt Solution) leaving 100 u1 of buffer after the final wash.
A stock solution of 1 mg/ml fluo-3 is made in 10% pluronic acid DMSO. To load
the
cells with fluo-3, 50 u1 of 12 ug/ml fluo-3 is added to each well. The plate
is incubated at
37~C in a CO2 incubator for 60 min. The plate is washed four times in the
Biotek washer
with HBSS leaving 100 u1 of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are
re-
suspended to 2-SxIO~ cells/ml with HBSS in a 50-ml conical tube. 4 u1 of 1
mg/ml fluo-3
solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The
tube is then
placed in a 37~C water bath for 30-60 min. The cells are washed twice with
HBSS,
resuspended to 1 x 10~ cells/ml, and dispensed into a microplate, 100 ul/well.
The plate is
centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley
CellWash with
200 u1, followed by an aspiration step to 100 u1 final volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as
fluo-3.
The supernatant is added to the well, and a change in fluorescence is
detected.
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To measure the fluorescence of intracellular calcium, the FLIPR is set for the
following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4
second; (3)
Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and
(6) Sample
addition is 50 u1. Increased emission at 530 nm indicates an extracellular
signaling event
caused by the a molecule, either D-SLAM or a molecule induced by D-SLAM, which
has
resulted in an increase in the intracellular Ca++ concentration.
Example 20: High-Throughput Screening Assay: Identt; fying Tyrosine Kinase
Activity
The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane
and
cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group
are
receptors for a range of mitogenic and metabolic growth factors including the
PDGF, FGF,
EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large
family of
RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include
mainly
secreted small proteins, but also membrane-bound and extracellular matrix
proteins.
Activation of RPTK by ligands involves ligand-mediated receptor dimerization,
resulting in transphosphorylation of the receptor subunits and activation of
the cytoplasmic
tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated
tyrosine
kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor
linked and cytosolic
protein tyrosine kinases, such as the Jak family, members of which mediate
signal
transduction triggered by the cytokine superfamily of receptors (e.g., the
Interleukins,
Interferons, GM-CSF, and Leptin).
Because of the wide range of known factors capable of stimulating tyrosine
kinase
activity, identifying whether D-SLAM or a molecule induced by D-SLAM is
capable of
activating tyrosine kinase signal transduction pathways is of interest.
Therefore, the
following protocol is designed to identify such molecules capable of
activating the tyrosine
kinase signal transduction pathways.
Seed target cells (e.g., primary keratinocytes) at a density of approximately
25,000
cells per well in a 96 well Loprodyne Silent Screen Plates purchased from
Nalge Nunc
(Naperville, IL). The plates are sterilized with two 30 minute rinses with
100% ethanol,
rinsed with water and dried overnight. Some plates are coated for 2 hr with
100 ml of cell
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culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50
mg/ml), all of which
can be purchased from Sigma Chemicals (St. Louis, MO) or 10% Matrigel
purchased from
Becton Dickinson (Bedford,MA), or calf serum, rinsed with PBS and stored at
4~C. Cell
growth on these plates is assayed by seeding 5,000 cells/well in growth medium
and indirect
quantitation of cell number through use of alamarBlue as described by the
manufacturer
Alamar Biosciences, Inc. (Sacramento, CA) after 48 hr. Falcon plate covers
#3071 from
Becton Dickinson (Bedford,MA) are used to cover the Loprodyne Silent Screen
Plates.
Falcon Microtest III cell culture plates can also be used in some
proliferation experiments.
To prepare extracts, A431 cells are seeded onto the nylon membranes of
Loprodyne
plates (20,000/200m1/well) and cultured overnight in complete medium. Cells
are quiesced
by incubation in serum-free basal medium for 24 hr. After 5-20 minutes
treatment with EGF
(60ng/ml) or 50 u1 of the supernatant produced in Example 12, the medium was
removed and
100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCI, 1 % Triton X-
100, 0.1
SDS, 2 mM Na3V04, 2 mM Na4P2O7 and a cocktail of protease inhibitors (#
1836170)
obtained from Boeheringer Mannheim (Indianapolis, IN) is added to each well
and the plate
is shaken on a rotating shaker for 5 minutes at 4oC. The plate is then placed
in a vacuum
transfer manifold and the extract filtered through the 0.45 mm membrane
bottoms of each
well using house vacuum. Extracts are collected in a 96-well catch/assay plate
in the bottom
of the vacuum manifold and immediately placed on ice. To obtain extracts
clarified by
centrifugation, the content of each well, after detergent solubilization for 5
minutes, is
removed and centrifuged for 15 minutes at 4~C at 16,000 x g.
Test the filtered extracts for levels of tyrosine kinase activity. Although
many
methods of detecting tyrosine kinase activity are known, one method is
described here.
Generally, the tyrosine kinase activity of a supernatant is evaluated by
determining its
ability to phosphorylate a tyrosine residue on a specific substrate (a
biotinylated peptide).
Biotinylated peptides that can be used for this purpose include PSK1
(corresponding to amino
acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to
amino acids 1-
17 of gastrin). Both peptides are substrates for a range of tyrosine kinases
and are available
from Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in
order.
First, add 10u1 of SuM Biotinylated Peptide, then 10u1 ATP/Mg2+ (SmM ATP/SOmM
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MgCl2), then 10u1 of 5x Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40
mM beta
glycerophosphate, 1mM EGTA, 100mM MgCl2, 5 mM MnCl2, 0.5 mg/ml BSA), then Sul
of
Sodium Vanadate(1mM), and then Sul of water. Mix the components gently and
preincubate
the reaction mix at 30~C for 2 min. Initial the reaction by adding l0ul of the
control enzyme
or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120mm
EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of
reaction
mixture to a microtiter plate (MTP) module and incubating at 37~C for 20 min.
This allows
the streptavadin coated 96 well plate to associate with the biotinylated
peptide. Wash the
MTP module with 300u1/well of PBS four times. Next add 75 ul of anti-
phospotyrosine
antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(O.Su/ml)) to
each well and
incubate at 37~C for one hour. Wash the well as above.
Next add 100u1 of peroxidase substrate solution (Boehringer Mannheim) and
incubate
at room temperature for at least 5 mins (up to 30 min). Measure the absorbance
of the sample
at 405 nm by using ELISA reader. The level of bound peroxidase activity is
quantitated
using an ELISA reader and reflects the level of tyrosine kinase activity.
Example 21: High-Throughput Screening Assay Identifying Phosphorylation
Activity
As a potential alternative and/or compliment to the assay of protein tyrosine
kinase
activity described in Example 20, an assay which detects activation
(phosphorylation) of
major intracellular signal transduction intermediates can also be used. For
example, as
described below one particular assay can detect tyrosine phosphorylation of
the Erk-1 and
Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK,
p38 MAP,
Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK,
Tec,
and Janus, as well as any other phosphoserine, phosphotyrosine, or
phosphothreonine
molecule, can be detected by substituting these molecules for Erk-1 or Erk-2
in the following
assay.
Specifically, assay plates are made by coating the wells of a 96-well ELISA
plate with
O.lml of protein G (lug/ml) for 2 hr at room temp, (RT). The plates are then
rinsed with PBS
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and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then
treated with 2
commercial monoclonal antibodies (100ng/well) against Erk-1 and Erk-2 (1 hr at
RT) (Santa
Cruz Biotechnology). (To detect other molecules, this step can easily be
modified by
substituting a monoclonal antibody detecting any of the above described
molecules.) After
3-5 rinses with PBS, the plates are stored at 4~C until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and
cultured
overnight in growth medium. The cells are then starved for 48 hr in basal
medium (DMEM)
and then treated with EGF (6ng/well) or 50 u1 of the supernatants obtained in
Example 12 for
5-20 minutes. The cells are then solubilized and extracts filtered directly
into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed.
As a
positive control, a commercial preparation of MAP kinase ( l Ong/well) is used
in place of
A431 extract. Plates are then treated with a commercial polyclonal (rabbit)
antibody
(lug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1
and Erk-2
kinases ( 1 hr at RT). This antibody is biotinylated by standard procedures.
The bound
polyclonal antibody is then quantitated by successive incubations with
Europium-streptavidin
and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument
(time-
resolved fluorescence). An increased fluorescent signal over background
indicates a
phosphorylation by D-SLAM or a molecule induced by D-SLAM.
Example 22: Method of Determining Alterations in the D-SLAM Gene
RNA isolated from entire families or individual patients presenting with a
phenotype
of interest (such as a disease) is be isolated. cDNA is then generated from
these RNA
samples using protocols known in the art. (See, Sambrook.) The cDNA is then
used as a
template for PCR, employing primers surrounding regions of interest in SEQ ID
NO:1.
Suggested PCR conditions consist of 35 cycles at 95~C for 30 seconds; 60-120
seconds at 52-
58~C; and 60-120 seconds at 70~C, using buffer solutions described in
Sidransky, D., et al.,
Science 252:706 ( 1991 ).
PCR products are then sequenced using primers labeled at their 5' end with T4
polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre
Technologies). The
intron-exon borders of selected exons of D-SLAM is also determined and genomic
PCR
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products analyzed to confirm the results. PCR products harboring suspected
mutations in D-
SLAM is then cloned and sequenced to validate the results of the direct
sequencing.
PCR products of D-SLAM are cloned into T-tailed vectors as described in
Holton,
T.A. and Graham, M.W., Nucleic Acids Research, 19:1156 (1991) and sequenced
with T7
polymerise (United States Biochemical). Affected individuals are identified by
mutations in
D-SLAM not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining
alterations in
the D-SLAM gene. Genomic clones isolated according to Example 2 are nick-
translated with
digoxigenindeoxy-uridine 5'-triphosphate (Boehringer Manheim), and FISH
performed as
described in Johnson, Cg. et al., Methods Cell Biol. 35:73-99 (1991).
Hybridization with the
labeled probe is carried out using a vast excess of human cot-1 DNA for
specific
hybridization to the D-SLAM genomic locus.
Chromosomes are counterstained .with 4,6-diamino-2-phenylidole and propidium
iodide, producing a combination of C- and R-bands. Aligned images for precise
mapping are
obtained using a triple-band filter set (Chroma Technology, Brattleboro, VT)
in combination
with a cooled charge-coupled device camera (Photometrics, Tucson, AZ) and
variable
excitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech. Appl.,
8:75 (1991).)
Image collection, analysis and chromosomal fractional length measurements are
performed
using the ISee Graphical Program System. (Inovision Corporation, Durham, NC.)
Chromosome alterations of the genomic region of D-SLAM (hybridized by the
probe) are
identified as insertions, deletions, and translocations. These D-SLAM
alterations are used as
a diagnostic marker for an associated disease.
Example 23: Method of Detecting Abnormal Levels of D-SLAM in a Biological
Sample
D-SLAM polypeptides can be detected in a biological sample, and if an
increased or
decreased level of D-SLAM is detected, this polypeptide is a marker for a
particular
phenotype. Methods of detection are numerous, and thus, it is understood that
one skilled in
the art can modify the following assay to fit their particular needs.
For example, antibody-sandwich ELISAs are used to detect D-SLAM in a sample,
preferably a biological sample. Wells of a microtiter plate are coated with
specific antibodies
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to D-SLAM, at a final concentration of 0.2 to 10 ug/ml. The antibodies are
either
monoclonal or polyclonal and are produced by the method described in Example
11. The
' wells are blocked so that non-specific binding of D-SLAM to the well is
reduced.
The coated wells are then incubated for > 2 hours at RT with a sample
containing D-
SLAM. Preferably, serial dilutions of the sample should be used to validate
results. The
plates are then washed three times with deionized or distilled water to remove
unbounded D-
SLAM.
Next, 50 u1 of specific antibody-alkaline phosphatase conjugate, at a
concentration of
25-400 ng, is added and incubated for 2 hours at room temperature. The plates
are again
washed three times with deionized or distilled water to remove unbounded
conjugate.
Add 75 u1 of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate
(NPP) substrate solution to each well and incubate 1 hour at room temperature.
Measure the
reaction by a microtiter plate reader. Prepare a standard curve, using serial
dilutions of a
control sample, and plot D-SLAM polypeptide concentration on the X-axis (log
scale) and
fluorescence or absorbance of the Y-axis (linear scale). Interpolate the
concentration of the
D-SLAM in the sample using the standard curve.
Example 24: Formulating a Polypeptide
The D-SLAM composition will be formulated and dosed in a fashion consistent
with
good medical practice, taking into account the clinical condition of the
individual patient
(especially the side effects of treatment with the D-SLAM polypeptide alone),
the site of
delivery, the method of administration, the scheduling of administration, and
other factors
known to practitioners. The "effective amount" for purposes herein is thus
determined by
such considerations.
As a general proposition, the total pharmaceutically effective amount of D-
SLAM
administered parenterally per dose will be in the range of about lug/kg/day to
10 mg/kg/day
of patient body weight, although, as noted above, this will be subject to
therapeutic
discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most
preferably for
humans between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, D-
SLAM is typically administered at a dose rate of about 1 ug/kg/hour to about
50 ug/kg/hour,
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either by 1-4 injections per day or by continuous subcutaneous infusions, for
example, using
a mini-pump. An intravenous bag solution may also be employed. The length of
treatment
needed to observe changes and the interval following treatment for responses
to occur
appears to vary depending on the desired effect.
Effective dosages of the compositions of the present invention to be
administered may
be determined through procedures well known to those in the art which address
such
parameters as biological half-life, bioavailability, and toxicity. Such
determination is well
within the capability of those skilled in the art, especially in light of the
detailed disclosure
provided herein.
Bioexposure of an organism to D-SLAM polypeptide during therapy may also play
an
important role in determining a therapeutically and/or pharmacologically
effective dosing
regime. Variations of dosing such as repeated administrations of a relatively
low dose of D-
SLAM polypeptide for a relatively long period of time may have an effect which
is
therapeutically and/or pharmacologically distinguishable from that achieved
with repeated
administrations of a relatively high dose of D-SLAM for a relatively short
period of time.
Using the equivalent surface area dosage conversion factors supplied by
Freireich, E.
J., et al. (Cancer Chemotherapy Reports 50(4):219-44 ( 1966)), one of ordinary
skill in the art
is able to conveniently convert data obtained from the use of D-SLAM in a
given
experimental system into an accurate estimation of a pharmaceutically
effective amount of D-
SLAM polypeptide to be administered per dose in another experimental system.
Experimental data obtained through the administration of D-SLAM may converted
through
the conversion factors supplied by Freireich, et al., to accurate estimates of
pharmaceutically
effective doses of D-SLAM in rat, monkey, dog, and human. The following
conversion table
(Table III) is a summary of the data provided by Freireich, et al. Table III
gives approximate
factors for converting doses expressed in terms of mg/kg from one species to
an equivalent
surface area dose expressed as mg/kg in another species tabulated.
Table III. Equivalent Surface Area Dosage Conversion Factors.
--TO--
Mouse Rat Monkey Dog Human
--FROM-- (20g) ( 1500 (3.Skg) (8kg) (60k~)
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Mouse 1 1/2 1/4 1/6 1/12
Rat 2 1 1/2 1/4 1/7
Monkey 4 2 1 3/5 1/3
Dog 6 4 5/3 1 1/2
Human 12 7 3 2 1
Thus, for example, using the conversion factors provided in Table III, a dose
of 50
mg/kg in the mouse converts to an appropriate dose of 12.5 mg/kg in the monkey
because (50
mg/kg) x (1/4) = 12.5 mg/kg. As an additional example, doses of 0.02, 0.08,
0.8, 2, and 8
mg/kg in the mouse equate to effect doses of 1.667 micrograms/kg, 6.67
micrograms/kg, 66.7
micrograms/kg, 166.7 micrograms/kg, and 0.667 mg/kg, respectively, in the
human.
Pharmaceutical compositions containing D-SLAM are administered orally,
rectally,
parenterally, intracistemally, intravaginally, intraperitoneally, topically
(as by powders,
ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal
spray. In one
embodiment, "pharmaceutically acceptable carrier" means a non-toxic solid,
semisolid or
liquid filler, diluent, encapsulating material or formulation auxiliary of any
type. In a specific
embodiment, "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 humans. Nonlimiting
examples of
suitable pharmaceutical carriers according to this embodiment are provided in
"Remington's
Pharmaceutical Sciences" by E.W. Martin, and include 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 be employed as liquid carriers,
particularly for injectable
solutions. 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.
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The term "parenteral" as used herein refers to modes of administration which
include
intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular
injection and infusion.
In a preferred embodiment, D-SLAM compositions of the invention (including
polypeptides, polynucleotides, and antibodies, and agonists and/or antagonists
thereof) are
administered subcutaneously.
In another preferred embodiment, D-SLAM compositions of the invention
(including
polypeptides, polynucleotides, and antibodies, and agonists and/or antagonists
thereof) are
administered intravenously.
D-SLAM compositions of the invention are also suitably administered by
sustained-
release systems. Suitable examples of sustained-release compositions include
suitable
polymeric materials (such as, for example, semi-permeable polymer matrices in
the form of
shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials
(for example as
an emulsion in an acceptable oil) or ion exchange resins, and sparingly
soluble derivatives
(such as, for example, a sparingly soluble salt).
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP
58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al.,
Biopolymers 22:547-556 ( 1983)), poly (2- hydroxyethyl methacrylate) (R.
Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105
(1982)),
ethylene vinyl acetate (R. Langer et al., Id.) or poly-D- (-)-3-hydroxybutyric
acid (EP
133,988).
Sustained-release compositions also include liposomally entrapped compositions
of
the invention (see generally, Langer, 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. 317 -327 and 353-365 (1989)). Liposomes containing
D-SLAM
polypeptide my be prepared by methods known per se: DE 3,218,121; Epstein et
al., Proc.
Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad.
Sci. (USA)
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;
Japanese
Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.
Ordinarily,
the liposomes are of the small (about 200-800 Angstroms) unilamellar type in
which the lipid
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content is greater than about 30 mol. percent cholesterol, the selected
proportion being
adjusted for the optimal D-SLAM polypeptide therapy.
In another embodiment systained release compositions of the invention include
crystal
formulations known in the art.
In yet an additional embodiment, the compositions of the invention are
delivered by
way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201
(1987);
Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.
321:574 (1989)).
Other controlled release systems are discussed in the review by Langer
(Science
249:1527-1533 (1990)).
For parenteral administration, in one embodiment, D-SLAM is formulated
generally
by mixing it at the desired degree of purity, in a unit dosage injectable form
(solution,
suspension, or emulsion),: with a pharmaceutically acceptable carrier, i.e.,
one that is non
toxic to recipients at the dosages and concentrations employed and is
compatible with other
ingredients of the formulation. For example, the formulation preferably does
not include
oxidizing agents and other compounds that are known to be deleterious to
polypeptides.
Generally, the formulations are prepared by contacting D-SLAM uniformly and
intimately with liquid carriers or finely divided solid carriers or both.
Then, if necessary, the
product is shaped into the desired formulation. Preferably the carrier is a
parenteral carrier,
more preferably a solution that is isotonic with the blood of the recipient.
Examples of such
carrier vehicles include water, saline, Ringer's solution, and dextrose
solution. Non-aqueous
vehicles such as fixed oils and ethyl oleate are also useful herein, as well
as liposomes.
The carrier suitably contains minor amounts of additives such as substances
that
enhance isotonicity and chemical stability. Such materials are non-toxic to
recipients at the
dosages and concentrations employed, and include buffers such as phosphate,
citrate,
succinate, acetic acid, and other organic acids or their salts; antioxidants
such as ascorbic
acid; low molecular weight (less than about ten residues) polypeptides, e.g.,
polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic
polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic
acid, aspartic
acid, or arginine; monosaccharides, disaccharides, and other carbohydrates
including
cellulose or its derivatives, glucose, manose, or dextrins; chelating agents
such as EDTA;
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sugar alcohols such as mannitol or sorbitol; counterions such as sodium;
and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
D-SLAM is typically formulated in such vehicles at a concentration of about
0.1
mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be
understood
that the use of certain of the foregoing excipients, carriers, or stabilizers
will result in the
formation of polypeptide salts.
D-SLAM used for therapeutic administration can be sterile. Sterility is
readily
accomplished by filtration through sterile filtration membranes (e.g., 0.2
micron membranes).
Therapeutic polypeptide compositions generally are placed into a container
having a sterile
access port, for example, an intravenous solution bag or vial having a stopper
pierceable by a
hypodermic injection needle.
D-SLAM polypeptides ordinarily will be stored in unit or multi-dose
containers, for
example, sealed ampoules or vials, as an aqueous solution or as a lyophilized
formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml vials are
filled with 5 ml
of sterile-filtered 1 % (w/v) aqueous D-SLAM polypeptide solution, and the
resulting mixture
is lyophilized. The infusion solution is prepared by reconstituting the
lyophilized D-SLAM
polypeptide using bacteriostatic Water-for-Injection.
The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of
the invention. Associated with such containers) can be a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
or biological
products, which notice reflects approval by the agency of manufacture, use or
sale for human
administration. In addition, D-SLAM may be employed in conjunction with other
therapeutic
compounds.
The compositions of the invention may be administered alone or in combination
with
other therapeutic agents. Therapeutic agents that may be administered in
combination with
the compositions of the invention, include but not limited to, members of the
TNF family
(e.g., Neutrokine-Alpha and/or Neutrokine-AlphaSV (International Application
No.
PCT/US96/17957) or antagonists thereof), chemotherapeutic agents, antibiotics,
steroidal and
non-steroidal anti-inflammatories, conventional immunotherapeutic agents,
cytokines and/or
growth factors. Combinations may be administered either concomitantly, e.g.,
as an
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admixture, separately but simultaneously or concurrently; or sequentially.
This includes
presentations in which the combined agents are administered together as a
therapeutic
mixture, and also procedures in which the combined agents are administered
separately but
simultaneously, e.g., as through separate intravenous lines into the same
individual.
Administration "in combination" further includes the separate administration
of one of the
compounds or agents given first, followed by the second.
In another specific embodiment, compositions of the invention are used in
combination with PNEUMOVAX-23T"" to treat, prevent, and/or diagnose infection
and/or any
disease, disorder, and/or condition associated therewith. In one embodiment,
compositions of
the invention are used in combination with PNEUMOVAX-23T"~ to treat, prevent,
and/or
diagnose any Gram positive bacterial infection and/or any disease, disorder,
and/or condition
associated therewith. In another embodiment, compositions of the invention are
used in
combination with PNEUMOVAX-23T"~ to treat, prevent, and/or diagnose infection
and/or any
disease, disorder, and/or condition associated with one or more members of the
genus
Enterococcus and/or the genus Streptococcus. In another embodiment,
compositions of the
invention are used in any combination with PNEUMOVAX-23T"" to treat, prevent,
and/or
diagnose infection and/or any disease, disorder, and/or condition associated
with one or more
members of the Group B streptococci. In another embodiment, compositions of
the invention
are used in combination with PNEUMOVAX-23T"" to treat, prevent, and/or
diagnose infection
and/or any disease, disorder, and/or condition associated with Streptococcus
pneumoniae.
The compositions of the invention may be administered alone or in combination
with
other therapeutic agents, including but not limited to, chemotherapeutic
agents, antibiotics,
antivirals, steroidal and non-steroidal anti-inflammatories, conventional
immunotherapeutic
agents and cytokines. Combinations may be administered either concomitantly,
e.g., as an
admixture, separately but simultaneously or concurrently; or sequentially.
This includes
presentations in which the combined agents are administered together as a
therapeutic
mixture, and also procedures in which the combined agents are administered
separately but
simultaneously, e.g., as through separate intravenous lines into the same
individual.
Administration "in combination" further includes the separate administration
of one of the
compounds or agents given first, followed by the second.
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In a specific embodiment, the compositions of the invention are administered
in
combination with antagonists of co-activators of B cells (e.g., Neutrokine-
Alpha and/or
Neutrokine-AlphaSV (International Application No. PCT/LTS96/17957), BLyS,
BAFF,
TALL-1, THANK, and/or zTNF4). In a further embodiment, antagonists may
include, but are
not limited to, antibodies directed to and/or the soluble extracellular
portion of the Neutokine-
Alpha and/or Neutrokine-AIphaSV receptor, the BLyS receptor, the BAFF
receptor, the
TALL-1 receptor, the THANK receptor, TACI, and/or BCMA.
In one embodiment, the compositions of the invention are administered in
combination with other members of the TNF family. TNF, TNF-related or TNF-like
molecules that may be administered with the compositions of the invention
include, but are
not limited to, soluble forms of 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), endokine-alpha
(International Publication No. WO 98/07880), Neutrokine-Alpha and/or
Neutrokine-AlphaSV
(International Application No. PCT/US96/17957), TR6 (International Publication
No. WO
98/30694), OPG, OX40, 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),
TRS
(International Publication No. WO 98/30693), TR6 (International Publication
No. WO
98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9
(International
Publication No. WO 98/56892), TR10 (International Publication No. WO
98/54202), 312C2
(International Publication No. WO 98/06842), and TR12, and soluble forms
CD154, CD70,
and CD 153.
In a preferred embodiment, the compositions of the invention are administered
in
combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g.,
AVRENDT~~),
biologically active fragments, variants, or derivatives of CD40L, anti-CD40L
antibodies
(e.g,. agonistic or antagonistic antibodies), and/or anti-CD40 antibodies
(e.g, agonistic or
antagonistic antibodies).
In another embodiment, compositions of the invention are administered in
combination with an anticoagulant. Anticoagulants that may be administered
with the
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compositions of the invention include, but are not limited to, heparin,
warfarin, and aspirin.
In a specific embodiment, compositions of the invention are administered in
combination
with heparin and/or warfarin. In another specific embodiment, compositions of
the invention
are administered in combination with warfarin. In another specific embodiment,
compositions of the invention are administered in combination with warfarin
and aspirin. In
another specific embodiemtn, compositions of the invention are administered in
combination
with heparin. In another specific embodiemtn, compositions of the invention
are
administered in combination with heparin and aspirin.
In another embodiment, compositions of the invention are administered in
combination with an agent that suppresses the production of anticardiolipin
antibodies. In
specific embodiments, the polynucleotides of the invention are administered in
combination
with an agent that blocks and/or reduces the ability of anticardiolipin
antibodies to bind
phospholipid-binding plasma protein beta 2-glycoprotein I (b2GPI).
In certain embodiments, compositions of the invention are administered in
combination with antiretroviral agents, nucleoside reverse transcriptase
inhibitors, non-
nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
Nucleoside reverse
transcriptase inhibitors that may be administered in combination with the
compositions of
the invention, include, but are not limited to, RETROVIRT"~ (zidovudine/AZT),
VIDEXT"'
(didanosine/ddI), HIVIDT"~ (zalcitabine/ddC), ZERITT"~ (stavudine/d4T),
EPIVIRT""
(lamivudine/3TC), and COMBIVIRT"' (zidovudine/lamivudine). Non-nucleoside
reverse
transcriptase inhibitors that may be administered in combination with the
compositions of
the invention, include, but are not limited to, VIRAMUNET"" (nevirapine),
RESCRIPTORT""
(delavirdine), and SUSTIVAT"~ (efavirenz). Protease inhibitors that may be
administered in
combination with the compositions of the invention, include, but are not
limited to,
CRIXIVANT"" (indinavir), NORVIRT~~ (ritonavir), INVIRASET"" (saquinavir), and
VIRACEPTT"~ (nelfinavir). In a specific embodiment, antiretroviral agents,
nucleoside
reverse transcriptase inhibitors, non-nucleoside reverse transcriptase
inhibitors, and/or
protease inhibitors may be used in any combination with compositions of the
invention to
treat, prevent, and/or diagnose AIDS and/or to treat, prevent, and/or diagnose
HIV infection.
In other embodiments, compositions of the invention may be administered in
combination with anti-opportunistic infection agents. Anti-opportunistic
agents that may be
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administered in combination with the compositions of the invention, include,
but are not
limited to, TRIMETHOPRIM-SULFAMETHOXAZOLET"~, DAPSONET"",
PENTAMIDINETM, ATOVAQUONET"", ISONIAZIDr"', RIFAMPINT"', PYRAZINAMIDETM,
ETHAMBUTOLT"~, RIFABUTINT"", CLARITHROMYCINT"", AZITHROMYCINT"~,
GANCICLOVIRT"~, FOSCARNETT"", CIDOFOVIRT"", FLUCONAZOLET"",
ITRACONAZOLET"", KETOCONAZOLET"~, ACYCLOVIRT"", FAMCICOLVIRT~~,
PYRIMETHAMINET"~, LEUCOVORINT"", NEUPOGENT"~ (filgrastim/G-CSF), and
LEUKINET"~ (sargramostim/GM-CSF). In a specific embodiment, compositions of
the
invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLET"~,
DAPSONET"~, PENTAMIDINET"", and/or ATOVAQUONET"~ to prophylactically treat,
prevent, and/or diagnose an opportunistic Pneumocystis carinii pneumonia
infection. In
another specific embodiment, compositions of the invention are used in any
combination with
ISONIAZIDT"~, RIFAMPINT~~, PYRAZINAMIDET"~, and/or ETHAMBUTOLT"" to
prophylactically treat, prevent, and/or diagnose an opportunistic
Mycobacterium avium
complex infection. In another specific embodiment, compositions of the
invention are used
in any combination with RIFABUTINT"~, CLARITHROMYCINT"~, and/or
AZITHROMYCINT"" to prophylactically treat, prevent, and/or diagnose an
opportunistic
Mycobacterium tuberculosis infection. In another specific embodiment,
compositions of the
invention are used in any combination with GANCICLOVIRT"~, FOSCARNETT"~,
and/or
CIDOFOVIRT"~ to prophylactically treat, prevent, and/or diagnose an
opportunistic
cytomegalovirus infection. In another specific embodiment, compositions of the
invention
are used in any combination with FLUCONAZOLET"~, ITRACONAZOLET"", and/or
KETOCONAZOLET"~ to prophylactically treat, prevent, and/or diagnose an
opportunistic
fungal infection. In another specific embodiment, compositions of the
invention are used in
any combination with ACYCLOVIRT"" and/or FAMCICOLVIRT"' to prophylactically
treat,
prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or
type II infection.
In another specific embodiment, compositions of the invention are used in any
combination
with PYRIMETHAMINET"' and/or LEUCOVORINT"" to prophylactically treat, prevent,
and/or diagnose an opportunistic Toxoplasma gondii infection. In another
specific
embodiment, compositions of the invention are used in any combination with
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LEUCOVORINTM and/or NEUPOGENT"~ to prophylactically treat, prevent, and/or
diagnose
an opportunistic bacterial infection.
In a further embodiment, the compositions of the invention are administered in
combination with an antiviral agent. Antiviral agents that may be administered
with the
compositions of the invention include, but are not limited to, acyclovir,
ribavirin,
amantadine, and remantidine.
In a further embodiment, the compositions of the invention are administered in
combination with an antibiotic agent. Antibiotic agents that may be
administered with the
compositions of the invention include, but are not limited to, amoxicillin,
aminoglycosides,
beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,
cephalosporins,
ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides,
metronidazole,
penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,
trimethoprim,
trimethoprim-sulfamthoxazole, and vancomycin.
Conventional nonspecific immunosuppressive agents, that may be administered in
combination with the compositions of the invention include, but are not
limited to, steroids,
cyclosporine, cyclosporine analogs cyclophosphamide, cyclophosphamide IV,
methylprednisolone, prednisolone, azathioprine, FK-506, 15-deoxyspergualin,
and other
immunosuppressive agents that act by suppressing the function of responding T
cells.
In specific embodiments, compositions of the invention are administered in
combination with immunosuppressants. Immunosuppressants preparations that may
be
administered with the compositions of the invention include, but are not
limited to,
ORTHOCLONET"' (OKT3), SANDIMMUNET~~/NEORALT"'/SANGDYAT"" (cyclosporin),
PROGRAFT"" (tacrolimus), CELLCEPTT"' (mycophenolate), Azathioprine,
glucorticosteroids,
and RAPAMUNET~~ (sirolimus). In a specific embodiment, immunosuppressants may
be
used to prevent rejection of organ or bone marrow transplantation.
In a preferred embodiment, the compositions of the invention are administered
in
combination with steroid therapy. Steroids that may be administered in
combination with
the compositions of the invention, include, but are not limited to, oral
corticosteroids,
prednisone, and methylprednisolone (e.g., IV methylprednisolone). In a
specific
embodiment, compositions of the invention are administered in combination with
prednisone. In a further specific embodiment, the compositions of the
invention are
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administered in combination with prednisone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the compositions of the
invention
and prednisone are those described herein, and include, but are not limited
to, azathioprine,
cylophosphamide, and cyclophosphamide IV. In a another specific embodiment,
compositions of the invention are administered in combination with
methylprednisolone. In
a further specific embodiment, the compositions of the invention are
administered in
combination with methylprednisolone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the compositions of the
invention
and methylprednisolone are those described herein, and include, but are not
limited to,
azathioprine, cylophosphamide, and cyclophosphamide IV.
In a preferred embodiment, the compositions of the invention are administered
in
combination with an antimalarial. Antimalarials that may be administered with
the
compositions of the invention include, but are not limited to,
hydroxychloroquine,
chloroquine, and/or quinacrine.
In a preferred embodiment, the compositions of the invention are administered
in
combination with an NSAID.
In a nonexclusive embodiment, the compositions of the invention are
administered in
combination with one, two, three, four, five, ten, or more of the following
drugs: NRD-101
(Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium
(Monsanto),
mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly),
atreleuton
(Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470
(Takeda),
CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431
(KS
Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522
(Japan
Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelone mesylate
(Warner-
Lambert), soluble TNF receptor 1 (synergen; Amgen), IPR-6001 (Institute for
Pharmaceutical Research), trocade (Hoffman-La Roche), EF-5 (Scotia
Pharmaceuticals),
BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim), LeukoVax
(Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), and butixocort propionate
(WarnerLambert).
In a preferred embodiment, the compositions of the invention are administered
in
combination with one, two, three, four, five or more of the following drugs:
methotrexate,
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sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine,
azathioprine,
an antimalarial drug (e.g., as described herein), cyclophosphamide,
chlorambucil, gold,
ENBRELT"" (Etanercept), anti-TNF antibody, LJP 394 (La Jolla Pharmaceutical
Company,
San Diego, California), and prednisolone.
In a more preferred embodiment, the compositions of the invention are
administered
in combination with an antimalarial, methotrexate, anti-TNF antibody,
ENBRELT"' and/or
suflasalazine. In one embodiment, the compositions of the invention are
administered in
combination with methotrexate. In another embodiment, the compositions of the
invention
are administered in combination with anti-TNF antibody. In another embodiment,
the
compositions of the invention are administered in combination with
methotrexate and anti-
TNF antibody. In another embodiment, the compositions of the invention are
administered in
combination with suflasalazine. In another specific embodiment, the
compositions of the
invention are administered in combination with methotrexate, anti-TNF
antibody, and
suflasalazine. In another embodiment, the compositions of the invention are
administered in
combination ENBRELT"". In another embodiment, the compositions of the
invention are
administered in combination with ENBRELT"~ and methotrexate. In another
embodiment, the
compositions of the invention are administered in combination with ENBRELT~~,
methotrexate and suflasalazine. In another embodiment, the compositions of the
invention
are administered in combination with ENBRELT"", methotrexate and
suflasalazine. In other
embodiments, one or more antimalarials is combined with one of the above-
recited
combinations. In a specfic embodiment, the compositions of the invention are
administered
in combination with an antimalarial (e.g., hydroxychloroquine), ENBRELT~",
methotrexate
and suflasalazine. In another specfic embodiment, the compositions of the
invention are
administered in combination with an antimalarial (e.g., hydroxychloroquine),
sulfasalazine,
anti-TNF antibody, and methotrexate.
In an additional embodiment, compositions of the invention are administered
alone or
in combination with one or more intravenous immune globulin preparations.
Intravenous
immune globulin preparations that may be administered with the compositions of
the
invention include, but not limited to, GAMMART"", IVEEGAMT~~,
SANDOGLOBULINT"",
GAMMAGARD S/DT"', and GAMIMUNET"". In a specific embodiment, compositions of
the
232
administered in combination
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invention are administered in combination with intravenous immune globulin
preparations in
transplantation therapy (e.g., bone marrow transplant).
In a further embodiment, the compositions of the invention are administered in
combination with an antibiotic agent. Antibiotic agents that may be
administered with the
compositions of the invention include, but are not limited to, tetracycline,
metronidazole,
amoxicillin, beta-lactamases, aminoglycosides, macrolides, quinolones,
fluoroquinolones,
cephalosporins, erythromycin, ciprofloxacin, and streptomycin.
In an additional embodiment, the compositions of the invention are
administered
alone or in combination with an anti-inflammatory agent. Anti-inflammatory
agents that may
be administered with the compositions of the invention include, but are not
limited to,
glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic
acid
derivatives, arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids,
arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid
derivatives,
thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-
hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome,
difenpiramide, ditazol,
emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol,
paranyline,
perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In another embodiment, compostions of the invention are administered in
combination with a chemotherapeutic agent. Chemotherapeutic agents that may be
administered with the compositions of the invention include, but are not
limited to, antibiotic
derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);
antiestrogens
(e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,
floxuridine,
interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-
thioguanine);
cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine
arabinoside,
cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,
busulfan, cis-platin,
and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine
phosphate
sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol
diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives
(e.g.,
mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and
combinations (e.g., bethamethasone sodium phosphate); and others (e.g.,
dicarbazine,
asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and
etoposide).
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In a specific embodiment, compositions of the invention are administered in
combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone) or
any combination of the components of CHOP. In another embodiment, compositions
of the
invention are administered in combination with Rituximab. In a further
embodiment,
compositions of the invention are administered with Rituxmab and CHOP, or
Rituxmab and
any combination of the components of CHOP.
In an additional embodiment, the compositions of the invention are
administered in
combination with cytokines. Cytokines that may be administered with the
compositions of
the invention include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4,
ILS, IL6, IL7,
IL 10, IL 12, IL 13, IL 15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma,
TNF-alpha,
and TNF-beta. In another embodiment, compositions of the invention may be
administered
with any interleukin, including, but not limited to, IL-lalpha, IL-lbeta, IL-
2, IL-3, IL,-4, IL-
5 , IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13 , IL-14, IL-15, IL-16,
IL-17, IL-18 , IL-
19, IL-20, IL-21, and IL-22. In preferred embodiments, the compositions of the
invention
are administered in combination with IL4 and IL10. Both IL4 and IL10 have been
observed
by the inventors to enhance D-SLAM-mediated B cell proliferation.
In an additional embodiment, the compositions of the invention are
administered with
a chemokine. In another embodiment, the compositions of the invention are
administered
with chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory
protein-4. In a
preferred embodiment, the compositions of the invention are administered with
chemokine
beta-8.
In an additional embodiment, the compositions of the invention are
administered in
combination with an IL-4 antagonist. IL-4 antagonists that may be administered
with the
compositions of the invention include, but are not limited to: soluble IL-4
receptor
polypeptides, multimeric forms of soluble IL-4 receptor polypeptides; anti-IL-
4 receptor
antibodies that bind the IL-4 receptor without transducing the biological
signal elicited by IL
4, anti-IL4 antibodies that block binding of IL-4 to one or more IL-4
receptors, and muteins
of IL-4 that bind IL-4 receptors but do not transduce the biological signal
elicited by IL-4.
Preferably, the antibodies employed according to this method are monoclonal
antibodies
(including antibody fragments, such as, for example, those described herein).
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In an additional embodiment, the compositions of the invention are
administered in
combination with hematopoietic growth factors. Hematopoietic growth factors
that may be
administered with the compositions of the invention include, but are not
limited to,
LEUKINET"' (SARGRAMOSTIMTM) and NEUPOGENT~~ (FILGRASTIMTM).
In an additional embodiment, the compositions of the invention are
administered in
combination with angiogenic proteins. Angiogenic proteins that may be
administered with
the compositions of the invention include, but are not limited to, Glioma
Derived Growth
Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet
Derived
Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110;
Platelet
Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-
282317;
Placental Growth Factor (P1GF), as disclosed in International Publication
Number WO
92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et al.,
Growth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in
International
Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A),
as
disclosed in European Patent Number EP-506477; Vascular Endothelial Growth
Factor-2
(VEGF-2), as disclosed in International Publication Number WO 96/39515;
Vascular
Endothelial Growth Factor B-186 (VEGF-B 186), as disclosed in International
Publication
Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as
disclosed in
International Publication Number WO 98/02543; Vascular Endothelial Growth
Factor-D
(VEGF-D), as disclosed in International Publication Number WO 98/07832; and
Vascular
Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number
DE19639601. The above mentioned references are incorporated herein by
reference herein.
In an additional embodiment, the compositions of the invention are
administered in
combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may
be
administered with the compositions of the invention include, but are not
limited to, FGF-l,
FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-
12,
FGF-13, FGF-14, and FGF-15.
In additional embodiments, the compositions of the invention are administered
alone
or in combination with other therapeutic or prophylactic regimens, including
but not limited
to, radiation therapy. Such combinatorial therapy may be administered
sequentially and/or
concomitantly.
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Example 25: Method of Treating Decreased Levels of D-SLAM
The present invention relates to a method for treating an individual in need
of a
decreased level of D-SLAM activity in the body comprising, administering to
such an
individual a composition comprising a therapeutically effective amount of D-
SLAM
antagonist. Preferred antagonists for use in the present invention are D-SLAM-
specific
antibodies.
Moreover, it will be appreciated that conditions caused by a decrease in the
standard
or normal expression level of D-SLAM in an individual can be treated by
administering D
SLAM, preferably in the secreted form. Thus, the invention also provides a
method of
treatment of an individual in need of an increased level of D-SLAM polypeptide
comprising
administering to such an individual a pharmaceutical composition comprising an
amount of
I S D-SLAM to increase the activity level of D-SLAM in such an individual.
For example, a patient with decreased levels of D-SLAM polypeptide receives a
daily
dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably,
the polypeptide
is in the secreted form. The exact details of the dosing scheme, based on
administration and
formulation, are provided in Example 24.
Example 26: Method of Treating Increased Levels of D-SLAM
The present invention also relates to a method for treating an individual in
need of an
increased level of D-SLAM activity in the body comprising administering to
such an
individual a composition comprising a therapeutically effective amount of D-
SLAM or an
agonist thereof.
Antisense technology is used to inhibit production of D-SLAM. This technology
is
one example of a method of decreasing levels of D-SLAM polypeptide, preferably
a secreted
form, due to a variety of etiologies, such as cancer.
For example, a patient diagnosed with abnormally increased levels of D-SLAM is
administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and
3.0 mg/kg day
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for 21 days. This treatment is repeated after a 7-day rest period if the
treatment was well
tolerated. The formulation of the antisense polynucleotide is provided in
Example 24.
Example 27: Method of Treatment Using Gene Therapy - Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of
expressing
D-SLAM polypeptides, onto a patient. Generally, fibroblasts are obtained from
a subject by
skin biopsy. The resulting tissue is placed in tissue-culture medium and
separated into small
pieces. Small chunks of the tissue are placed on a wet surface of a tissue
culture flask,
approximately ten pieces are placed in each flask. The flask is turned upside
down, closed
tight and left at room temperature over night. After 24 hours at room
temperature, the flask is
inverted and the chunks of tissue remain fixed to the bottom of the flask and
fresh media
(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.
The flasks are
then incubated at 37~C for approximately one week.
At this time, fresh media is added and subsequently changed every several
days.
After an additional two weeks in culture, a monolayer of fibroblasts emerge.
The monolayer
is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 ( 1988)), flanked by the long
terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI
and HindIII
and subsequently treated with calf intestinal phosphatase. The linear vector
is fractionated on
agarose gel and purified, using glass beads.
The cDNA encoding D-SLAM can be amplified using PCR primers which correspond
to the 5' and 3' end sequences respectively as set forth in Example 1.
Preferably, the 5' primer
contains an EcoRI site and the 3' primer includes a HindIII site. Equal
quantities of the
Moloney murine sarcoma virus linear backbone and the amplified EcoRI and
HindIII
fragment are added together, in the presence of T4 DNA ligase. The resulting
mixture is
maintained under conditions appropriate for ligation of the two fragments. The
ligation
mixture is then used to transform bacteria HB 101, which are then plated onto
agar containing
kanamycin for the purpose of confirming that the vector contains properly
inserted D-SLAM.
The amphotropic pA317 or GP+am 12 packaging cells are grown in tissue culture
to
confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf
serum
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(CS), penicillin and streptomycin. The MSV vector containing the D-SLAM gene
is then
added to the media and the packaging cells transduced with the vector. The
packaging cells
now produce infectious viral particles containing the D-SLAM gene(the
packaging cells are
now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is
harvested from a 10 cm plate of confluent producer cells. The spent media,
containing the
infectious viral particles, is filtered through a millipore filter to remove
detached producer
cells and this media is then used to infect fibroblast cells. Media is removed
from a sub-
confluent plate of fibroblasts and quickly replaced with the media from the
producer cells.
This media is removed and replaced with fresh media. If the titer of virus is
high, then
virtually all fibroblasts will be infected and no selection is required. If
the titer is very low,
then it is necessary to use a retroviral vector that has a selectable marker,
such as neo or his.
Once the fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine
whether D-SLAM protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone
or after
having been grown to confluence on cytodex 3 microcarrier beads.
Example 28: Gene Therapy Using Endogenous D-SLAM Gene
Another method of gene therapy according to the present invention involves
operably associating the endogenous D-SLAM sequence with a promoter via
homologous
recombination as described, for example, in U.S. Patent No. 5,641,670, issued
June 24, 1997;
International Publication No. WO 96/29411, published September 26, 1996;
International
Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc.
Natl. Acad. Sci.
USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989). This
method
involves the activation of a gene which is present in the target cells, but
which is not
expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and targeting
sequences, which are homologous to the 5' non-coding sequence of endogenous D-
SLAM,
flanking the promoter. The targeting sequence will be sufficiently near the 5'
end of D
SLAM so the promoter will be operably linked to the endogenous sequence upon
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homologous recombination. The promoter and the targeting sequences can be
amplified
using PCR. Preferably, the amplified promoter contains distinct restriction
enzyme sites on
the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence
contains the same
restriction enzyme site as the 5' end of the amplified promoter and the 5' end
of the second
targeting sequence contains the same restriction site as the 3' end of the
amplified promoter.
The amplified promoter and the amplified targeting sequences are digested with
the
appropriate restriction enzymes and subsequently treated with calf intestinal
phosphatase.
The digested promoter and digested targeting sequences are added together in
the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation
of the two fragments. The construct is size fractionated on an agarose gel
then purified by
phenol extraction and ethanol precipitation.
In this Example, the polynucleotide constructs are administered as naked
polynucleotides via electroporation. However, the polynucleotide constructs
may also be
administered with transfection-facilitating agents, such as liposomes, viral
sequences, viral
particles, precipitating agents, etc. Such methods of delivery are known in
the art.
Once the cells are transfected, homologous recombination will take place which
results in the promoter being operably linked to the endogenous D-SLAM
sequence. This
results in the expression of D-SLAM in the cell. Expression may be detected by
immunological staining, or any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is placed
in DMEM + 10% fetal calf serum. Exponentially growing or early stationary
phase
fibroblasts are trypsinized and rinsed from the plastic surface with nutrient
medium. An
aliquot of the cell suspension is removed for counting, and the remaining
cells are subjected
to centrifugation. The supernatant is aspirated and the pellet is resuspended
in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCI, 5 mM KCI, 0.7 mM Na,
HPO~, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated,
and the cells
resuspended in electroporation buffer containing 1 mg/ml acetylated bovine
serum albumin.
The final cell suspension contains approximately 3X l Ofi cells/ml.
Electroporation should be
performed immediately following resuspension.
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Plasmid DNA is prepared according to standard techniques. For example, to
construct a plasmid for targeting to the D-SLAM locus, plasmid pUC 18 (MBI
Fermentas,
Amherst, NY) is digested with HindIII. The CMV promoter is amplified by PCR
with an
XbaI site on the 5' end and a BamHI site on the 3'end. Two D-SLAM non-coding
sequences
are amplified via PCR: one D-SLAM non-coding sequence (D-SLAM fragment 1) is
amplified with a HindIII site at the 5' end and an Xba site at the 3'end; the
other D-SLAM
non-coding sequence (D-SLAM fragment 2) is amplified with a BamHI site at the
5'end and a
HindIII site at the 3'end. The CMV promoter and D-SLAM fragments are digested
with the
appropriate enzymes (CMV promoter - XbaI and BamHI; D-SLAM fragment 1 - XbaI;
D-
SLAM fragment 2 - BamHI) and ligated together. The resulting ligation product
is digested
with HindIII, and ligated with the HindIII-digested pUCl8 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-
Rad).
The final DNA concentration is generally at least 120 pg/ml. 0.5 ml of the
cell suspension
(containing approximately 1.S.X10~ cells) is then added to the cuvette, and
the cell
suspension and DNA solutions are gently mixed. Electroporation is performed
with a
Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 ~F and
250-300 V,
respectively. As voltage increases, cell survival decreases, but the
percentage of surviving
cells that stably incorporate the introduced DNA into their genome increases
dramatically.
Given these parameters, a pulse time of approximately 14-20 mSec should be
observed.
Electroporated cells are maintained at room temperature for approximately 5
min, and
the contents of the cuvette are then gently removed with a sterile transfer
pipette. The cells
are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a
10 cm dish and incubated at 37~C. The following day, the media is aspirated
and replaced
with 10 ml of fresh media and incubated for a further 16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or
after having
been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now
produce the
protein product. The fibroblasts can then be introduced into a patient as
described above.
Example 29: Method of Treatment Using Gene Therapy - In Vivo
Another aspect of the present invention is using in vivo gene therapy methods
to treat,
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diagnose, detect, and/or prevent disorders, diseases and conditions. The gene
therapy method
relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA
or RNA)
D-SLAM sequences into an animal to increase or decrease the expression of the
D-SLAM
polypeptide. The D-SLAM polynucleotide may be operatively linked to a promoter
or any
other genetic elements necessary for the expression of the D-SLAM polypeptide
by the target
tissue. Such gene therapy and delivery techniques and methods are known in the
art, see, for
example, WO 90/11092, WO 98/11779; U.S. Patent NO. 5,693,622, 5,705,151,
5,580,859;
Tabata H. et al. (1997) Cardiovasc. Res. 35(3):470-479, Chao J et al. (1997)
Pharmacol. Res.
35(6):517-522, Wolff J.A. (1997) Neuromuscul. Disord. 7(5):314-318, Schwartz
B. et al.
(1996) Gene Ther. 3(5):405-411, Tsurumi Y. et al. (1996) Circulation
94(12):3281-3290
(incorporated herein by reference).
The D-SLAM polynucleotide constructs may be delivered by any method that
delivers injectable materials to the cells of an animal, such as, injection
into the interstitial
space of tissues (heart, muscle, skin, lung, liver, intestine and the like).
The D-SLAM
polynucleotide constructs can be delivered in a pharmaceutically acceptable
liquid or aqueous
carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences that are free
from any delivery vehicle that acts to assist, promote, or facilitate entry
into the cell,
including viral sequences, viral particles, liposome formulations, lipofectin
or precipitating
agents and the like. However, the D-SLAM polynucleotides may also be delivered
in
liposome formulations (such as those taught in Felgner P.L. et al. ( 1995)
Ann. NY Acad. Sci.
772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be
prepared by
methods well known to those skilled in the art.
The D-SLAM polynucleotide vector constructs used in the gene therapy method
are
preferably constructs that will not integrate into the host genome nor will
they contain
sequences that allow for replication. Any strong promoter known to those
skilled in the art
can be used for driving the expression of DNA. Unlike other gene therapies
techniques, one
major advantage of introducing naked nucleic acid sequences into target cells
is the transitory
nature of the polynucleotide synthesis in the cells. Studies have shown that
non-replicating
DNA sequences can be introduced into cells to provide production of the
desired polypeptide
for periods of up to six months.
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The D-SLAM polynucleotide construct can be delivered to the interstitial space
of
tissues within the an animal, including of muscle, skin, brain, lung, liver,
spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,
and connective
tissue. Interstitial space of the tissues comprises the intercellular fluid,
mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers in the
walls of vessels or
chambers, collagen fibers of fibrous tissues, or that same matrix within
connective tissue
ensheathing muscle cells or in the lacunae of bone. It is similarly the space
occupied by the
plasma of the circulation and the lymph fluid of the lymphatic channels.
Delivery to the
interstitial space of muscle tissue is preferred for the reasons discussed
below. They may be
conveniently delivered by injection into the tissues comprising these cells.
They are
preferably delivered to and expressed in persistent, non-dividing cells which
are
differentiated, although delivery and expression may be achieved in non-
differentiated or less
completely differentiated cells, such as, for example, stem cells of blood or
skin fibroblasts.
In vivo muscle cells are particularly competent in their ability to take up
and express
polynucleotides.
For the naked D-SLAM polynucleotide injection, an effective dosage amount of
DNA
or RNA will be in the range of from about 0.05 g/kg body weight to about 50
mg/kg body
weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg
and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan
of ordinary
skill will appreciate, this dosage will vary according to the tissue site of
injection. The
appropriate and effective dosage of nucleic acid sequence can readily be
determined by those
of ordinary skill in the art and may depend on the condition being treated and
the route of
administration. The preferred route of administration is by the parenteral
route of injection
into the interstitial space of tissues. However, other parenteral routes may
also be used, such
as, inhalation of an aerosol formulation particularly for delivery to lungs or
bronchial tissues,
throat or mucous membranes of the nose. In addition, naked D-SLAM
polynucleotide
constructs can be delivered to arteries during angioplasty by the catheter
used in the
procedure.
The dose response effects of injected D-SLAM polynucleotide in muscle in vivo
is
determined as follows. Suitable D-SLAM template DNA for production of mRNA
coding
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for D-SLAM polypeptide is prepared in accordance with a standard recombinant
DNA
methodology. The template DNA, which may be either circular or linear, is
either used as
naked DNA or complexed with liposomes. The quadriceps muscles of mice are then
injected
with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by
intraperitoneal
injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the
anterior thigh, and
the quadriceps muscle is directly visualized. The D-SLAM template DNA is
injected in 0.1
ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute,
approximately 0.5
cm from the distal insertion site of the muscle into the knee and about 0.2 cm
deep. A suture
is placed over the injection site for future localization, and the skin is
closed with stainless
steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are
prepared by
excising the entire quadriceps. Every fifth 15 um cross-section of the
individual quadriceps
muscles is histochemically stained for D-SLAM protein expression. A time
course for D-
SLAM protein expression may be done in a similar fashion except that
quadriceps from
different mice are harvested at different times. Persistence of D-SLAM DNA in
muscle
following injection may be determined by Southern blot analysis after
preparing total cellular
DNA and HIRT supernatants from injected and control mice. The results of the
above
experimentation in mice can be use to extrapolate proper dosages and other
treatment
parameters in humans and other animals using D-SLAM naked DNA.
Example 30: D-SLAM Transgenic Animals.
The D-SLAM polypeptides can also be expressed in transgenic animals.
Animals of any species, including, but not limited to, mice, rats, rabbits,
hamsters, guinea
pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys,
and chimpanzees may be used to generate transgenic animals. In a specific
embodiment,
techniques described herein or otherwise known in the art, are used to express
polypeptides of
the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene (i.e.,
polynucleotides of the invention) into animals to produce the founder lines of
transgenic
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animals. Such techniques include, but are not limited to, pronuclear
microinjection (Paterson
et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,
Biotechnology (NY)
11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and
Hoppe et al.,
U.S. Pat. No. 4,873,191 ( 1989)); retrovirus mediated gene transfer into germ
lines (Van der
Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts
or embryos;
gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (
1989));
electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814
(1983));
introduction of the polynucleotides of the invention using a gene gun (see,
e.g., Ulmer et al.,
Science 259:1.745 ( 1993); introducing nucleic acid constructs into embryonic
pleuripotent
stem cells and transferring the stem cells back into the blastocyst; and sperm-
mediated gene
transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such
techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989), which is
incorporated
by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones
containing
polynucleotides of the invention, for example, nuclear transfer into
enucleated oocytes of
nuclei from cultured embryonic, fetal, or adult cells induced to quiescence
(Campell et al.,
Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
The present invention provides for transgenic animals that carry the transgene
in all
their cells, as well as animals which carry the transgene in some, but not all
their cells, i.e.,
mosaic animals or chimeric. The transgene may be integrated as a single
transgene or as
multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-
tail tandems.
The transgene may also be selectively introduced into and activated in a
particular cell type
by following, for example, the teaching of Lasko et al. (Lasko et al., Proc.
Natl. Acad. Sci.
USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-
type specific
activation will depend upon the particular cell type of interest, and will be
apparent to those
of skill in the art. When it is desired that the polynucleotide transgene be
integrated into the
chromosomal site of the endogenous gene, gene targeting is preferred.
Briefly, when such a technique is to be utilized, vectors containing some
nucleotide
sequences homologous to the endogenous gene are designed for the purpose of
integrating,
via homologous recombination with chromosomal sequences, into and disrupting
the function
of the nucleotide sequence of the endogenous gene. The transgene may also be
selectively
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introduced into a particular cell type, thus inactivating the endogenous gene
in only that cell
type, by following, for example, the teaching of Gu et al. (Gu et al., Science
265:103-106
(1994)). The regulatory sequences required for such a cell-type specific
inactivation will
depend upon the particular cell type of interest, and will be apparent to
those of skill in the
art. The contents of each of the documents recited in this paragraph is herein
incorporated by
reference in its entirety.
Any of the D-SLAM polypeptides disclose throughout this application can be
used to
generate transgenic animals. For example, DNA encoding amino acids M1-K232 of
SEQ ID
N0:2 can be inserted into a vector containing a promoter, such as the actin
promoter, which
will ubiquitously express the inserted fragment. Primers that can be used to
generate such
fragments include a 5' primer containing a BamHI restriction site shown in
bold:
GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC(SEQ
ID NO: 22) and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:
24). This construct will express the predicted extracellular domain of D-SLAM
under the
control of the actin promoter for ubiquitous expression. The region of D-SLAM
included in
this construct extends from M1-K232 of SEQ ID N0:2.
Similarly, the DNA encoding the full length D-SLAM protein can also be
inserted
into a vector using the following primers: A 5' primer containing a BamHI
restriction site
shown in bold:
GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ
ID NO: 22) and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO:
23). Besides these two examples, other fragments of D-SLAM can also be
inserted into a
vector to create transgenics having ubiquitous expression.
Alternatively, polynucleotides of the invention can be inserted in a vector
which
controls tissue specific expression through a tissue specific promoter. For
example, a
construct having a transferrin promoter would express the D-SLAM polypeptide
in the liver
of transgenic animals. Therefore, DNA encoding amino acids M1-K232 of SEQ ID
N0:2
can be amplified using a 5' primer, having a BamHI restriction site shown in
bold:
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GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ
ID NO: 22), and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:
24).
Similarly, the DNA encoding the full length D-SLAM protein can also be
inserted
into a vector for tissue specific expression using the following primers: A 5'
primer
containing a BamHI restriction site shown in bold:
GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ
ID NO: 22) and a 3' primer, containing a Xba restriction site shown in bold:
GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO:
23).
Once transgenic animals have been generated, the expression of the recombinant
gene
may be assayed utilizing standard techniques. Initial screening may be
accomplished by
Southern blot analysis or PCR techniques to analyze animal tissues to verify
that integration
of the transgene has taken place. The level of mRNA expression of the
transgene in the
tissues of the transgenic animals may also be assessed using techniques which
include, but
are not limited to, Northern blot analysis of tissue samples obtained from the
animal, in situ
hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of
transgenic gene
expressing tissue may also be evaluated immunocytochemically or
immunohistochemically
using antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or
crossbred to produce colonies of the particular animal. Examples of such
breeding strategies
include, but are not limited to: outbreeding of founder animals with more than
one
integration site in order to establish separate lines; inbreeding of separate
lines in order to
produce compound transgenics that express the transgene at higher levels
because of the
effects of additive expression of each transgene; crossing of heterozygous
transgenic animals
to produce animals homozygous for a given integration site in order to both
augment
expression and eliminate the need for screening of animals by DNA analysis;
crossing of
separate homozygous lines to produce compound heterozygous or homozygous
lines; and
breeding to place the transgene on a distinct background that is appropriate
for an
experimental model of interest.
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Transgenic animals of the invention have uses which include, but are not
limited to,
animal model systems useful in elaborating the biological function of D-SLAM
polypeptides,
studying conditions and/or disorders associated with aberrant D-SLAM
expression, and in
screening for compounds effective in ameliorating such conditions and/or
disorders.
Example 31: D-SLAM Knock-Out Animals.
Endogenous D-SLAM gene expression can also be reduced by inactivating or
"knocking out" the D-SLAM gene and/or its promoter using targeted homologous
recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas &
Capecchi,
Cell 51:503-512 ( 1987); Thompson et al., Cell 5:313-321 ( 1989); each of
which is
incorporated by reference herein in its entirety). For example, a mutant, non-
functional
polynucleotide of the invention (or a completely unrelated DNA sequence)
flanked by DNA
homologous to the endogenous polynucleotide sequence (either the coding
regions or
regulatory regions of the gene) can be used, with or without a selectable
marker and/or a
negative selectable marker, to transfect cells that express polypeptides of
the invention in
vivo. In another embodiment, techniques known in the art are used to generate
knockouts in
cells that contain, but do not express the gene of interest. Insertion of the
DNA construct, via
targeted homologous recombination, results in inactivation of the targeted
gene. Such
approaches are particularly suited in research and agricultural fields where
modifications to
embryonic stem cells can be used to generate animal offspring with an inactive
targeted gene
(e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this
approach can
be routinely adapted for use in humans provided the recombinant DNA constructs
are directly
administered or targeted to the required site in vivo using appropriate viral
vectors that will be
apparent to those of skill in the art.
For example, a targeting vector can be created which incorporates fragments of
the
murine D-SLAM locus flanking a Neomyocin cassette. The two flanking regions
are
generated using the following primers. For the 5' ARM flanking region:
5' ARM primer F: 5'AGCCGGTACCTCCGATGTGCATAATCAGGCT 3' (SEQ ID
N0:27), with an Asp718 restriction site underlined; and
5' ARM primer R: S' GCTTGGCGCGCCCCTGGTTAGTCTGCCTATGTA 3' (SEQ ID
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