U.S. patent application number 11/048669 was filed with the patent office on 2005-06-09 for heterologous polypeptide of the tnf family.
Invention is credited to Ambrose, Christine, Cachero, Teresa G., Rennert, Paul, Thompson, Jeffrey S..
Application Number | 20050124543 11/048669 |
Document ID | / |
Family ID | 22665269 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124543 |
Kind Code |
A1 |
Rennert, Paul ; et
al. |
June 9, 2005 |
Heterologous polypeptide of the TNF family
Abstract
A newly identified heteromeric ligand of the Tumor Necrosis
Factor (TNF)-family, referred to hereinafter as "APBF" has been
identified.
Inventors: |
Rennert, Paul; (Millis,
MA) ; Thompson, Jeffrey S.; (Stoneham, MA) ;
Ambrose, Christine; (Reading, MA) ; Cachero, Teresa
G.; (Brookline, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
1251 AVENUE OF THE AMERICAS FL C3
NEW YORK
NY
10020-1105
US
|
Family ID: |
22665269 |
Appl. No.: |
11/048669 |
Filed: |
February 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11048669 |
Feb 1, 2005 |
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10214065 |
Aug 7, 2002 |
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6875846 |
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10214065 |
Aug 7, 2002 |
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PCT/US01/04121 |
Feb 8, 2001 |
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60181670 |
Feb 11, 2000 |
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Current U.S.
Class: |
424/85.1 ;
424/143.1; 514/19.3; 530/350; 530/388.22 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 38/00 20130101; A61P 35/00 20180101; C07K 14/52 20130101; A61P
37/06 20180101; A61P 43/00 20180101; A61P 19/04 20180101; A61P
37/02 20180101; A61P 19/02 20180101; A61P 11/00 20180101; C07K
14/70575 20130101; A61P 15/00 20180101; A61P 17/00 20180101; A61P
29/00 20180101; A61P 1/16 20180101; A61P 17/02 20180101 |
Class at
Publication: |
514/012 ;
424/143.1; 530/350; 530/388.22 |
International
Class: |
A61K 039/395; C07K
014/74; C07K 016/28 |
Claims
1-16. (canceled)
17. An antibody specific for APBF.
Description
RELATED APPLICATIONS
[0001] This is a continuation of PCT/US01/04121, filed on Feb. 8,
2001, which claims priority from U.S. provisional application Ser.
No. 60/181,670 filed on Feb. 11, 2000.
TECHNICAL FIELD
[0002] This invention relates, in part, to a newly identified
heteromeric ligand of the Tumor Necrosis Factor (TNF)-family,
referred to hereinafter as "APBF", its variants, derivatives,
agonists and antagonists; and uses thereof. In particular, the
invention relates to an APBF having a TNF-family member APRIL
subunit linked non-covalently to a TNF-family member BAFF
subunit.
BACKGROUND OF THE INVENTION
[0003] Tumor Necrosis Factor (TNF)-family members can best be
described as master switches in the immune system controlling both
cell survival and differentiation. Given the current progress in
manipulating members of the TNF-family for therapeutic benefit,
including anti-tumor activity as well as immune regulation and
inflammation, it is likely that members of this family will provide
unique means to control disease. The medical utility of the TNF
ligands and antagonists to the ligands has been shown for several
systems. Most notable is TNF. TNF controls a wide array of immune
processes, including inducing acute inflammatory reactions, as well
as maintaining lymphoid tissue homeostasis. Because of the dual
role this cytokine can play in various pathological settings, both
agonist and antagonist reagents have been developed as modifers of
disease. For example TNF and LT.alpha. (which also signals through
the TNF receptors) have been used as a treatment for cancers,
especially those residing in peripheral sites, such as limb
sarcomas. In this setting direct signaling by the cytokine through
the receptor induces tumor cell death (Aggarwal and Natarajan,
1996. Eur Cytokine Netw 7:93-124). In immunological settings agents
which block TNF receptor signaling (eg., anti-TNF mAb, soluble
TNF-R fusion proteins) have been used to treat diseases like
rheumatoid arthritis and inflammatory bowel disease. In these
pathologies, TNF is acting to induce cell proliferation and
effector function, thereby exacerbating autoimmune disease. In this
setting blocking TNF binding to its receptor(s) has therapeutic
benefit (Beutler, 1999. J Rheumatol 26 Suppl 57:16-21).
[0004] A more recently discovered ligand/receptor system appears
amenable to similar manipulations. Lymphotoxin beta (LT.beta.), a
TNF family member which forms heterotrimers with LT.alpha., binds
to the LT.beta.-R. Some adenocarcinoma tumor cells which express
LT.beta.-R can be killed or differentiated when treated with an
agonistic anti-LT.beta.-R mAb (Browning et al., 1996. J Exp Med
183: 867-878). In immunological settings it has been shown that
anti-LT.alpha. mAb or soluble receptor fusion protein LT.beta.-R-Ig
can block the development of inflammatory bowel diseases, possibly
by influencing dendritic cell and T cell interaction (Mackay et
al., 1998. Gastroenterology 115:1464-1475).
[0005] In addition to the TNFR and LT.beta.-R systems, manipulation
of the TRAIL (Gura, 1997. Science 277: 768) and OPG (Simonet et al.
1997. Cell 89: 309-319) pathways may be therapeutically beneficial
in treating cancer and bone loss, respectively. Recently, through
database searches, there has been a number of newly described
members of the TNF family of ligands and receptors. In addition to
the number of new members, the complexity of the ligand/receptor
interactions has also increased. It is now apparent that the TNF
and LT systems are not unique in the ability of the ligand to
interact with more than one receptor. Among the ligands reported to
bind more than one receptor or receptor decoy are FasL, TRAIL,
RANKL, and LIGHT.
[0006] Thus, there is a clear need to identify and characterize
additional molecules which are members of the TNF family thereby
providing additional means of controlling disease and manipulating
the immune system.,
SUMMARY OF THE INVENTION
[0007] The present invention relates to the identification of a
newly discovered heteromer in the TNF-family, APBF, its nucleotide
sequences, its protein sequences and resulting polynucleotides,
polypeptides as well as to its soluble form; receptor to the APBF
and antibodies specific for APBF and its receptor; and uses
therefrom.
[0008] The invention relates to an isolated polypeptide comprising
an APRIL subunit linked via a non-covalent interaction to a BAFF
subunit. In one aspect the invention is directed to an isolated
polypeptide comprising an APRIL subunit selected from the group
consisting of human APRIL, partial human APRIL, murine APRIL or
partial murine APRIL, or amino acid substitution variants thereof;
linked via non-covalent interaction to a BAFF subunit selected from
the group consisting of human BAFF, partial human BAFF, murine BAFF
or partial murine BAFF, or amino acid substitution variants
thereof. In preferred embodiments, the partial BAFF or APRIL
polypeptides are soluble portions of the polypeptides.
[0009] In preferred embodiments of the invention, the heterologous
polypeptide comprises more than one APRIL subunit, and more
preferably two APRIL subunits, linked non-covalently to a BAFF
subunit. In alternative embodiments, the heterologous polypeptide
comprises more than one BAFF subunit, and more preferably two BAFF
subunits, linked l0 non-covalently to an APRIL subunit. Thus, in
preferred embodiments, the present invention is directed to
heterologous polypeptide trimers of BAFF and APRIL subunits, in
which the ratio of APRIL to BAFF subunits is 2:1, or alternatively
1:2.
[0010] The present invention also relates to therapeutic methods
utilizing the heteromers of the invention. One aspect of the
invention relates to methods of inhibiting B-cell, T-cell or tumor
cell growth in an animal by administering a therapeutically
effective amount of a composition selected from the group
consisting of an isolated APBF molecule or active fragment thereof,
a recombinant APBF molecule or active fragment thereof, and an
antibody specific for APBF or an active fragment thereof. Another
aspect of the invention relates to methods of stimulating B-cell or
T-cell growth in an animal by administering a therapeutically
effective amount of a composition selected from the group
consisting of an isolated APBF molecule or active fragment thereof,
a recombinant APBF molecule or active fragment thereof, and an
antibody specific for APBF or an active fragment thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The following drawings depicts certain embodiments of the
invention. They are illustrative only and do not limit the
invention otherwise disclosed herein.
[0012] FIG. 1a shows the amino acid sequence of human APRIL (SEQ ID
NO: 2). The predicted transmembrane region (TM, boxed), the
potential N-linked glycosylation site (star) and the N-terminus of
the recombinant soluble APRIL sequences are indicated. FIG. 1b
shows the DNA sequence encoding human APRIL (SEQ ID NO.: 1), the
amino acid sequence of human APRIL (SEQ ID NO.: 2) is shown in FIG.
1c, the DNA sequence encoding mouse APRIL (SEQ ID NO.: 3) is shown
in FIG. 1d, and the amino acid sequence of mouse APRIL (SEQ ID NO.:
4) is shown in FIG. 1e.
[0013] FIG. 2a shows the DNA sequence encoding human BAFF (SEQ ID
NO.: 5), the amino acid sequence of human BAFF (SEQ ID NO.: 6) is
shown in FIG. 2b, the DNA sequence encoding mouse BAFF (SEQ ID NO.:
7) is shown in FIG. 2c-2d, and the amino acid sequence of mouse
BAFF (SEQ ID NO.: 8) is shown in FIG. 2d. Amino acids 1 to 46 from
SEQ ID NO.: 6 represent the intracellular domain, amino acids 47 to
72 from SEQ ID NO.: 6 represent the transmembrane domain and amino
acids 73 to 285 from SEQ ID NO.: 6 represent the extracellular
domain.
[0014] FIG. 3 shows a comparison of two western blots from cells
co-transfected with various APRIL and BAFF encoding plasmids. The
detection reagent used in Panel A is an anti-FLAG antibody. The
detection reagent used in Panel B is an anti-BAFF antibody.
[0015] FIG. 4 shows a western blot of the immunoprecipiations of
conditioned media from cells co-transfected with plasmids encoding
various soluble APRIL and soluble BAFF proteins and
immunoprecipitated with an anti-FLAG-tagged antibody. The detection
reagent for the western blot is an anti-myc tagged antibody.
DETAILED DESCRIPTION
[0016] Definitions
[0017] The term "APBF" or "APBF ligand" when used herein
encompasses any native or recombinantly produced polypeptide having
an APRIL subunit linked via a non-covalent interaction to a BAFF
subunit. APBF may be isolated from a variety of sources, such as
from murine or human tissue types or from other sources, or
prepared by recombinant or synthetic methods. A large number of
analytical biochemistry methods, known to those of skill in the
art, can be utilized to determine the stoichiometry of APBF, its
variants and derivatives. For example, cation exchange
chromatography can be used to determine which of the various
stoichiometric forms are present in the preparation derived from
affinity columns. Also gel chromatography of the purified fractions
will show the molecular weights of each form. The molecular weights
of APRIL and BAFF are known. For example, the molecular weight of
full length human BAFF, amino acids 1-285, is predicted to be 34.2
kDa for each polypeptide. The molecular weight of soluble human
BAFF, amino acids A132-285, is predicted to be 18.2 kDa per
polypeptide. The molecular weight of full length human APRIL, amino
acids 1-250, is predicted to be 30.0 kDa for each polypeptide. The
molecular weight of soluble human APRIL, amino acids A105-250, is
predicted to be 17.5 kDa per polypeptide. Stoichiometric
combinations contemplated in the present invention include the
following formula, X APRIL: Y BAFF, where X and Y are integers
greater than or equal to one. It is contemplated that the heteromer
may exist as a soluble molecule, wherein all subunits are of
soluble APRIL or BAFF polypeptides. It is further contemplated that
the heteromer may exist as a cell associated molecule, wherein at
least one of the subunits is the full length molecule containing a
transmembrane domain and the other subunit(s) may contain either
full length or soluble forms of APRIL or BAFF.
[0018] The term "APRIL subunit" when used herein encompasses any
native or recombinantly produced APRIL polypeptide. The APRIL
subunit may be isolated from a variety of sources, such as from
murine or human tissue types or from other sources, or prepared by
recombinant or synthetic methods. For example, an APRIL subunit can
have an amino acid sequence encoded by human APRIL (SEQ ID NO.: 1)
or murine APRIL (SEQ ID NO.: 3) and variants, derivatives and
unique fragments thereof. Specifically contemplated are human and
murine soluble construct forms of APRIL (see above, and SEQ ID
NOs.: 2 and SEQ ID NO.: 4) and variants, derivatives and unique
fragments thereof.
[0019] The term "BAFF subunit" when used herein encompasses any
native or recombinantly produced BAFF polypeptide. The BAFF subunit
may be isolated from a variety of sources, such as from murine or
human tissue types or from other sources, or prepared by
recombinant or synthetic methods. For example, a BAFF subunit can
have an amino acid sequence encoded by human BAFF (SEQ ID NO.: 5)
or murine BAFF (SEQ ID NO.: 7) and variants, derivatives and unique
fragments thereof. Specifically contemplated are human and murine
soluble construct forms of BAFF (see above, and SEQ ID NO.: 6 and
SEQ ID NOs.: 8) and variants, derivatives and unique fragments
thereof.
[0020] As defined herein, a "unique fragment" of a protein or
nucleic acid is a peptide or oligonucleotide of sufficient length
to have a sequence unique to a particular gene or polypeptide,
i.e., a sequence not shared by related or unrelated genes or
polypeptides. Thus, for example, a unique nucleic acid fragment
typically will have at least 16 nucleotide residues, and a unique
polypeptide fragment typically will have at least 6 amino acid
residues. Preferably, to ensure substantially unique occurrence in
a typical higher eukaryotic genome, a unique nucleic acid fragment
should have at least 20 nucleotide residues, and a unique
polypeptide fragment should have at least 8 amino acid
residues.
[0021] An "isolated" polypeptide, polynucleotide, protein,
antibody, or other substance refers to a preparation of the
substance devoid of at least some of the other components that may
also be present where the substance or a similar substance
naturally occurs or is initially obtained from. Thus, for example,
an isolated substance may be prepared by using a purification
technique to enrich it from a source mixture. Enrichment can be
measured on an absolute basis, such as weight per volume of
solution, or it can be measured in relation to a second,
potentially interfering substance present in the source mixture.
Increasing enrichments of the embodiments of this invention are
increasingly more preferred. Thus, for example, a 2-fold enrichment
is preferred, 10-fold enrichment is more preferred, 100-fold
enrichment is more preferred, 1000-fold enrichment is even more
preferred. A substance can also be provided in an isolated state by
a process of artificial assembly, such as by chemical synthesis or
recombinant expression. "Hybridization" is the noncovalent,
antiparallel bonding of complementary nucleic acid strands, in
which Watson-Crick base pairing is established. To ensure
specificity, hybridization should be carried out under stringent
conditions, defined herein as conditions of time, temperature,
probe length, probe and/or target concentration, osmotic strength,
pH, detergent, carrier nucleic acid, etc. that permit no more than
an occasional base-pairing mismatch within a probe/target duplex.
Highly stringent conditions exclude all but about one base pair
mismatch per kb of target sequence. Exemplary highly stringent
conditions involve hybridization to membrane immobilized target
nucleic acid at a temperature of 65.degree. C. in the presence of
0.5 m NaHPO.sub.4, 7% SDS, ImM EDTA, followed by washing at
68.degree. C. in the presence of 0.1.times.SSC, 0.1% SDS. Current
Protocols in Molecular Biology (1989), Ausubel et al., eds, Greene
Publishing and Wiley Interscience, New York, N.Y. In circumstances
where relatively infrequent mismatches, e.g., up to about ten
mismatches per kb of target, can be tolerated, moderately stringent
conditions may be used. For moderate stringency, probe/target
hybrids formed under the above conditions are washed at 42.degree.
C. in the presence of 0.2.times.SSC, 0.1% SDS.
[0022] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to, farm
animals, sport animals, pets, primates, mice and rats.
[0023] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an effective amount is an amount of APBF, variants and
derivatives of APBF and agonists and antagonists of APBF that is
sufficient to ameliorate, stabilize, or delay development of a
disease state associated with APBF. Particularly APBF-associated
tumors. Detection and measurement of these indicators of efficacy
are discussed below.
[0024] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, one or more of the following: alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, preventing spread (i.e., metastasis)
of disease, preventing occurrence or recurrence of disease, delay
or slowing of disease progression, amelioration of the disease
state, and remission (whether partial or total). Also encompassed
by "treatment" is a reduction of pathological consequences of an
APBF-associated tumor(s).
[0025] As used herein, the term "cancer" refers to any neoplastic
disorder, including such cellular disorders as, for example, renal
cell cancer, Kaposi's sarcoma, chronic leukemia, breast cancer,
sarcoma, ovarian carcinoma, rectal cancer, throat cancer, melanoma,
colon cancer, bladder cancer, mastocytoma, lung cancer, mammary
adenocarcinoma, pharyngeal squamous cell carcinoma, and
gastrointestinal or stomach cancer. Preferably, the cancer is
leukemia, mastocytoma, melanoma, lymphoma, mammary adenocarcinoma,
and pharyngeal squamous cell carcinoma.
[0026] To determine the "percent homology" of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino acid or nucleic acid sequence). The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are homologous at that position (i.e.,
as used herein amino acid or nucleic acid "homology" is equivalent
to amino acid or nucleic acid "identity"). The percent homology
between the two sequences is a function of the number of identical
positions shared by the sequences (i.e., % homology=# of identical
positions/total # of positions.times.100). The determination of
percent homology between two sequences can be accomplished using a
mathematical algorithim. A preferred, non-limiting example of a
mathematical algorithim utilized for the comparison of two
sequences is the algorithm of Karlin and Altschul (1990) Proc.
Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-77.
[0027] The invention encompasses all nucleic acids, peptides,
polynucleotides, polypeptides and proteins of the present invention
that can be produced, expressed, and/or manipulated by conventional
molecular engineering techniques such as the techniques set forth
in Current Protocols in Molecular Cloning, Ausubel et al., eds.
(1989), Greene Publishing and Wiley Interscience, New York, N.Y.
and in Sambrook et al. (1989), Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., and the teachings described and referenced in Watson
et al. (1992), Recombinant DNA 2nd ed., Scientific American Books
and W. H. Freeman & Co., New York, N.Y.
DESCRIPTION OF THE INVENTION
[0028] The present invention relates to a newly identified
heteromeric member of the TNF-family, APBF, wherein APBF comprises
an APRIL subunit linked non-covalently to a BAFF subunit.
[0029] APRIL, a TNF ligand known to have a role in inducing tumor
cell proliferation is described in detail in PCT publications
WO99/12965, WO97/33902, WO99/50416 and U.S. Provisional Application
Ser. No. 60/106,976, each incorporated by reference herein. It has
been shown that high levels of APRIL mRNA are detected in several
tumor cell lines, as well as in colon carcinomas, metastatic
lymphomas and thyroid tumors. Moreover, it has been shown that the
in vitro addition of recombinant APRIL stimulates the proliferation
of various cell lines. It is also recognized that in addition to
inducing tumor cell proliferation that APRIL may modulate a variety
of functions of immune system cells in vitro and in vivo (Hahne et
al., (1998) J. Exp. Med. 188 :1185-1190).
[0030] The second component of APBF, BAFF, has been shown to have a
role in inducing the proliferation of naive B cells and is
described in detail in PCT publications WO98/18921, WO98/27114, and
WO99/12964, each incorporated by reference herein. Like APRIL, BAFF
has also been shown to modulate a variety of functions of immune
system cells in vitro (Schneider et al., (1999) J. Exp. Med. 189:
1747-1756) and in vivo (Mackay et al., (1999) J. Exp.
Med.190:1697-1710; Moore et al., (1999) Science.285: 260-263).
[0031] To date, all known TNF-family members, with the exception of
the lymphotoxins, form homomers. It was therefore a surprising
discovery, as a result of the work described herein, to identify a
heteromeric polypeptide having an APRIL subunit linked
non-covalently to a BAFF subunit FIGS. 1 and 2 provides the full
length and partial nucleic acid and amino acid sequences of
mammalian APRIL and mammalian BAFF, respectively. The
intracellular, transmembrane, and extracellular domains are
identified, and a protease cleavage site is marked. N-terminal
amino acid sequence analysis of APRIL secreted into the media of
EBNA293 cells transfected with the full length murine APRIL cDNA
plasmid identified alanine at position 87 as the first amino acid
in the secreted form. Similar analysis of human BAFF overexpressed
in EBNA293 cells showed that alanine at position 134 (numbering of
amino acids corresponds to the naturally occuring human BAFF
sequence, as found for example, in Schneider et al. 1999 J. Exp.
Med 189: 1747-1756) was the first amino acid of the secreted form
through amino acid 285.
[0032] In one embodiment, APBF comprises an APRIL subunit derived
from a mammalian APRIL linked via a non-covalent interaction to a
BAFF subunit derived from a mammalian BAFF. It is contemplated that
subunits of APRIL and/or BAFF may remain cell membrane bound via
their transmembrane domains, and comprise part of a membrane-bound
APBF. Alternatively, the APBF may consist of the natively cleaved
forms of APRIL and BAFF extracellular domains, or fragments derived
from the natively cleaved forms. As illustrated in Example 1, when
FLAG-tagged soluble APRIL is co-expressed with full-length BAFF,
the soluble heteromeric complex is formed. This shows that the
full-length BAFF is readily cleaved and complexes with the
artificially generated soluble APRIL molecule. Alternatively,
Example 2 demonstrates that the complex can be formed when both
APRIL and BAFF are expressed as soluble molecules. This indicates
that the region between the transmembrane and receptor binding
domain (stalk) is not required for association. However, if one or
more subunits remains uncleaved then the complex will remain
tethered to the cell surface. Alternatively, the complex will be
secreted. Since additional modification may take place after
proteolytic cleavage from the cell surface, other subunit forms are
envisioned, for example, one or more subunits may consist of a
portion of the extracellular domain, as when the stalk portion
(before the first beta sheet) is shortened. Also, as APRIL and BAFF
contain glycosylation sites it is conceivable that one or more
subunits may be a glycosylated or differentially glycosylated. Such
modifications may depend on the cell in which the heteromer is
expressed.
[0033] As a result of the work described herein, in which we
identified APBF by co-expression and differentially tagging (see
Examples 1 and 2), we are able to produce and isolate APBF by any
of a number of techniques known to those of skill in the art,
including for example, affinity methods, as described for example,
in Example 3. Another example of a known method for isolation of
proteins include ion exchange chromatography. For example, APBF may
easily be separated by ion exchange chromatography based on the
widely ranging pI values for soluble human APRIL (ie: hAPRIL
pI=9.81) and soluble human BAFF (hBAFF pI=4.75). Heterocomplexes
commonly have pI values that are additive in nature.
Stoichiometrically different combinations of BAFF and APRIL
proteins would be expected to bind DEAE and S-sepharose columns
with significantly different affinities at a given pH.
Visualization of these heterocomplexes can also be done by
isoelectic focusing (IEF), followed by blotting and detection with
antibodies. IEF can also be used to isolate small amounts of
proteins. Since native IEF generally does not disrupt protein
function it may well present itself as a useful way to assay the
protein binding affinities cells and receptors and to evaluate
transfections for the level of the various heteromers produced.
Such methods are particularly useful in separating different
subunit stoichiometries which may be present after
co-transfection.
[0034] The invention further provides degenerate variant nucleic
acids that encode the SEQ ID NOS.: 2, 4, 6 and 8 polypeptides or a
unique fragment thereof. In yet further embodiments, the invention
provides nucleic acids encoding variant APBF polypeptides,
comprising amino acid sequences sharing at least 75% sequence
similarity with the SEQ ID NOs.: 2, 4, 6 and/or 8. Preferably,
these nucleic acids encode polypeptides sharing at least 80%, 85%,
90% or more preferably 95% amino acid sequence similarity with SEQ
ID NOs.: 2, 4, 6 and/or 8. The encoded variant polypeptides
comprise amino acid mutations (substitutions, deletions and/or
insertions) distributed in any random or non-random frequency
within SEQ ID NOs.: 2, 4, 6 and/or 8. "Similarity" as used herein
refers to the sum of aligned amino acid residues that are identical
to the residues of corresponding SEQ ID NOs.: 2, 4, 6 and 8 and
those that are allowed point mutations therefor. Moderate gaps
and/or insertions (e.g., less than about 50, preferably less than
about 15, more preferably less than about 5 amino acid residues) in
the aligned sequence are ignored for similarity calculation
purposes. Allowed point mutations are substitutions by amino acid
residues that are physically and/or functionally similar to the
corresponding aligned residues of SEQ ID NOs.: 2, 4, 6 and/or 8,
e.g., that have similar size, shape, hydrophilic or hydrophobic
character, charge and chemical properties.
[0035] It should be understood that the present invention provides
oligonucleotides that hybridize to any of the foregoing variant
APBF nucleic acids, i.e., to nucleic acids that encode polypeptides
comprising amino acid sequences that share at least 75% sequence
-similarity with SEQ ID NOs: 2, 4, 6 and/or 8. More particularly,
the invention provides olignucleotides that hybridize to one or
more unique fragments of nucleic acids encoding APBF. For
therapeutic purposes and/or for PCR investigative or diagnostic
purposes, the present oligonucleotides hybridize to a unique
fragment comprising 5' untranslated sequence, a transcription
initiation site, ORF or polypeptide coding sequence, intron-exon
boundary, polyadenylation site or 3' untranslated region of the
present APBF nucleic acids.
[0036] The invention also relates to heteromers formed with partial
sequences of human and murine APRIL and human and murine BAFF.
Preferably, these partial sequences comprise soluble forms of BAFF
and APRIL. Preferred partial human APRIL molecules include amino
acids A105 to L250, K110 to L250 and H115 to L250 of the
full-length human APRIL sequence. Preferred partial murine APRIL
molecules include amino acids A87 to L233 of the full-length murine
APRIL sequence. Preferred partial human BAFF sequences include
amino acids A134 to L285 and Q136 to L285 of the full-length human
BAFF sequence (see, Schneider et al. 1999, J.Exp. Med.
189:1747-1756, incorporated by reference herein).
[0037] Preferred partial sequences of human and murine APRIL and
BAFF also include splice variants of APRIL and BAFF. A preferred
partial human APRIL sequence includes a splice variant that is the
complete APRIL human sequence missing amino acids 113 to 128 (see,
Kelly et al. 2000, Can. Res. 60: 1021-1027, incorporated herein by
reference). Preferred partial human BAFF sequences include a splice
variant which is the full-length BAFF sequence missing amino acids
142 to 160 (see, WO 00/50597). Preferred partial murine BAFF
sequences include a splice variant which is full-length BAFF
sequence missing amino acids 166 to 184.
[0038] The invention also encompasses soluble secreted forms of
APBF. See Example 2. To create a soluble secreted form of APBF, one
would use techniques known to those of skill in the art, including
for example removing at the DNA level the N-terminus transmembrane
regions encoding either the APRIL and/or BAFF N-terminus
transmembrane regions, and some portion of the corresponding stalk
region, and replace these regions with a type I leader or
alternatively a type II leader sequence that will allow efficient
proteolytic cleavage in the chosen expression system. A skilled
artisan could vary the amount of the stalk region retained in the
secretion expression construct to optimize both receptor binding
properties and secretion efficiency. For example, the constructs
containing all possible stalk lengths, i.e. N-terminal truncations,
could be prepared such that proteins starting at amino acids
105-135, for APRIL and 134-164 for BAFF would result. The optimal
length stalk sequence would result from this type of analysis.
[0039] Isolated APBF can be used for a number of purposes, such as
the production of monoclonal or polyclonal antibodies, and
identification of novel modulators affecting biological function
(e.g., inhibitors), and identification of receptors interacting
with APBF.
[0040] As a result of the work described herein, antibodies
(polyclonal or monoclonal) specific for the identified APBF can be
produced, using known methods (See, for example, Antibodies: A
Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:
1988)). Such antibodies and host cells (i.e. hybridoma cells)
producing the antibodies are also the subject of the present
invention.
[0041] Antibody production involves administration of one or more
immunogenic doses of an APBF polypeptide preparation (whether
isolated or incorporated in a cell membrane) to an appropriate
non-human animal, such as a mouse, rat, rabbit, guinea pig, turkey,
goat, sheep, pig, or horse. To enhance immunogenicity, the
preparation can be emulsified with a conventional adjuvant, such as
Freund's complete or incomplete adjuvant. Routine monitoring of
serum immunoglobulins, using peripheral blood samples withdrawn at
appropriate intervals (e.g., seven to ten days) after an initial or
subsequent immunization, can be used to detect the onset and/or
maturation of a humoral immune response. Detection and, optionally,
quantitation, of immunoglobulins selectively reactive with an APBF
epitope can be achieved through any conventional technique, such as
ELISA, radioimmunoassay, Western blotting, or the like.
[0042] An immunoglobulin "selectively reactive with an APBF
epitope" has binding specificity for the recognized epitope such
that an antibody/epitope complex forms under conditions generally
permissive of the formation of such complexes (e.g., under
conditions of time, temperature, ionic strength, pH ionic or
nonionic detergent, carrier protein, etc.). Serial dilution
(titration) analysis by standard techniques is useful to estimate
the avidity of antibodies in the immune serum sample for one or
more epitopes unique to APBF. As defined herein, an "epitope unique
to APBF" is a unique, immunogenic fragment of the full-length APBF
polypeptide. A unique linear epitope typically ranges in size from
about ten to about twenty-five amino acid residues, and frequently
is about twelve to eighteen residues in length.
[0043] Immune serum having a high titer generally is preferred
herein. Serum having a half maximal avidity for a unique APBF
epitope of at least about 1:1000, preferably at least about
1:10,000, can be harvested in bulk for use as a source of
polyclonal antibody useful in the detection and/or quantitation of
APBF. Polyclonal immunoglobulins can, if desired, be enriched by
conventional fractionation of such serum, or can be isolated by
conventional immunoadsorbent techniques, e.g., using a Protein A or
Protein G chromatography resin. Immune, high titer murine, rat,
hamster or guinea pig serum alternatively is preferred herein for
the production and screening of hybridomas secreting monoclonal
antibodies selectively reactive with APBF. The present hybridomas
can be produced according to well-known, standard techniques. The
present monoclonal antibodies can be obtained from hybridoma
culture supernatant, or from conventionally produced ascites fluid,
and optionally isolated via immunoadsorbent chromatography or
another suitable separation technique prior to use as agents to
detect and/or quantitate APBF.
[0044] A preferred antibody, whether polyclonal or monoclonal, is
selectively reactive with a unique APBF epitope that is displayed
on the surface of or secreted from APBF expressing cells. The
preferred antibody accordingly can be used to detect and, if
desired, quantitate APBF expressing cells, e.g., normal or
transformed cells in a mammalian body tissue or biopsy sample
thereof. Specifically, the preferred antibody can be used to detect
APBF expressing cells whether such cells are host cells or
mammalian body tissue cells that aberrantly express APBF.
Advantageously, intact, e.g., living, cells that display a unique
APBF epitope can be detected by standard immunohistochemical,
radiometric imaging or flow cytometry techniques. The present
antibody can be used to detect and/or monitor APBF polypeptide
production. Thus, the antibody can be used to assess the natural
tissue-specific production of APBF, and thus to assess tissues
likely to give rise to carcinomas or sarcomas. In addition, the
present antibody can be used to monitor tumor biopsy samples to
provide information relevant to selecting or revising a course of
disease management, or to diagnosis, prognostication and/or staging
of any disease associated with an abnormality affecting APBF.
Furthermore, the present antibody can be used in a cell-sorting
procedure or other cell isolation procedure to generate a
substantially pure preparation of APBF expressing cells, or a cell
population substantially depleted of APBF expressing cells. Each of
the foregoing can be achieved through routine practice or
modification of well-known techniques, including but not limited to
the conjugation of a detectable moiety (e.g., a radionuclide,
fluorophore, chromophore, binding pair member, or enzyme) to the
APBF reactive antibody.
[0045] A hybridoma secreting an APBF reactive monoclonal antibody
of the present invention additionally provides a suitable source of
nucleic acid for the routine construction of a fusion polypeptide
comprising an antigen-binding fragment derived from the APBF
reactive antibody. The present fusion polypeptide can be prepared
by routine adaptation of conventional techniques described in
Deeley et al. (1996), U.S. Pat. No. 5,489,519 (incorporated herein
by reference). The fusion polypeptide can be a truncated
immunoglobulin, an immunoglobulin having a desired constant region
(e.g., IgG in lieu of IgM), or a "humanized" immunoglobulin having
an APBF reactive Fc region fused to a framework region of human
origin. Additional fusion polypeptides can comprise, in addition to
an APBF reactive antigen-binding fragment, a non-immunoglobulin
polypeptide such as a cytotoxic polypeptide (e.g., diphtheria
toxin, ricin) or a chemoattractant polypeptide that stimulates
immune effector cells (cytotoxic T cells, natural killer cells,
macrophages) to kill cells that display APBF. Standard techniques
well-known in the art can be used to produce appropriate
immunoglobulin fusion polypeptides of the present invention.
[0046] Various forms of antibodies can also be made using standard
recombinant DNA techniques. For example, humanization techniques
have been developed that render non-human Mabs less antigenic in
humans. Methods for humanizing Mabs by chimerisation procedures are
described in EP0120694, EP0125023, EP-A-0 171496, EP-A-0173 494 and
WO 86/01533, each incorporated by reference herein. Chimerisation
procedures generally involve preparing antibody having the variable
domains from a mouse MAb and the constant domains from a human
immunoglobulin. Alternatively, methods for humanizing Mabs by
CDR-grafting are described in EP-A-0239400 (Winter), WO90/07861
(Queen), WO91/09967 (CellTech), and WO91/09967 (CellTech),
incorporated by reference herein. CDR-grafting generally involves
grafting the complementarity determining regions (CDRs) of a mouse
MAb onto the framework regions of the variable domains of a human
immunoglobulin by site directed mutagenesis using long
oligonucleotides. In WO91/09967, the preparation of humanized
CDR-grafted antibody products which have specificity for TNF-alpha
is described. In particular WO91/09967 describes in Example 5,
preparation of specific humanized CDR grafted antibodies to human
TNF-alpha derived from murine anti-human TNF-alpha Mabs. Using any
of these known methods, therefore, antibodies specific for APBF can
be produced and isolated.
[0047] The polypeptides and methods disclosed herein enable the
identification of receptors which specifically interact with APBF
or fragments thereof. For example, the APBF receptor can be cloned
using any of the techniques known to those of skill in the art,
including for example, one or more of the following approaches.
[0048] For example, one can identify an APBF receptor using
expression cloning in mammalian cells. Specifically, a cDNA
expression library is generated from a cell line or cell population
shown to express the highest level of the receptor to the protein
of interest, i.e. APBF. This approach was shown for the leptin
receptor (Tartaglia et al., 1995 Cell 83: 1263-1271). The cDNA
library DNA is made as pools of 2-3,000 cDNAs and transfected into
an appropriate' cell line which does not express the receptor. A
plate assay format may be used to detect expression of the receptor
on the surface of the receptor negative cell line using purified
APBF. An antibody to one of the subunits or to an epitope tag is
used to detect the bound protein of interest, i.e. APBF and an
alkaline phosphatase conjugated secondary antibody and alkaline
phosphatase substrates are used to visualize the positive cells.
The plate wells are screened using a microscope. The complexity of
the cDNA pools from the positive wells are reduced and then
screened again. The screening continues until transfection of a
single cDNA produces a positive signal. The DNA of the cDNA is
sequenced and the predicted amino acid sequence analyzed for motifs
and structure consistent with members of the TNF receptor family.
Other expression cloning formats are available, for example, by
panning on ligand coating plates, or by sorting with tagged ligand
in FACS analyses.
[0049] In another approach, one can identify an APBF receptor using
direct DNA sequence analysis. Specifically, a directional cDNA
library is generated from a receptor positive cell line and the 5'
ends sequenced using ABI automated DNA sequencing technology to
determine the open reading frame. Programs to look at the cysteine
residue spacing, signal and transmembrane sequences can be employed
to identify potential TNF receptor family members. Full-length
clones will then be isolated, expressed and examined for the
ability to bind APBF. The library can also be subtracted with a
APBF receptor negative cell line to reduce the complexity of the
library.
[0050] In another approach, one can identify an APBF receptor by
examining known or orphan receptors. Specifically, purified APBF
can be used in FACS, immunoprecipitation, ELISA or Biacore assays
against a panel of orphan receptors and those that have known
ligands. A receptor for APBF will be positive in all of these
assays.
[0051] In yet another approach, one can identify an APBF receptor
using protein sequence analysis methods. Specifically, APBF can be
cross-linked to the surface of cells determined to be receptor
positive using standard reagents. The cross-linked complex can be
immunoprecipitated using an antibody to one of the subunits or an
epitope tag on the subunit. The complex can be separated on a SDS
polyacrylamide, blotted to a membrane and subjected to amino acid
analysis. Once amino acid sequencing reveals information about the
receptor, degenerate oligonucleotide probes can be synthesized and
used to screen a cDNA library made from a receptor positive cell
line.
[0052] Alternatively, one can identify an APBF receptor using known
biochemical approaches in combination with mass spectometry to
specifically identify masses and sequences. Three illustrative
examples are set forth below:
[0053] Strategy 1: Binding of [125I]-labelled APBF to different
cell lines. After choosing the appropriate cell line, crosslinking
of [125]I-APBF to the cells followed by polyacrylamide gel
electrophoresis (SDS-PAGE) and autoradiography will reveal a
receptor protein that will be analyze by mass spectrometry.
[0054] Strategy 2: Crosslinking of [125I]-labelled APBF to cell
lines, followed by immunoprecipitation using specific antibodies
and polyacrylamide gel electrophoresis.
[0055] Strategy 3. Choosing appropriate cell lines that bind APBF.
Adding purified APBF would allow the heteromer binding to the
receptor. Immunoprecipitation using specific antibodies to the
heteromer followed by SDS-PAGE and autoradiography will reveal
specific bands that will be isolated and analyzed by mass
spectrometry to identify the receptor/s.
[0056] The foregoing compositions can be used for a number of
purposes, including the assessment (e.g., for diagnostic purposes)
of abnormalities in the structure and/or expression of a cellular
APBF gene as well as diagnosis of conditions involving abnormally
high or low expression of APBF activities. For example, in
WO/99/12965, it was shown that while transcript of APRIL are of low
abundance in normal tissues, high levels of mRNA are detected in
several tumor cells lines. The expression and growth stimulating
effect of APRIL on tumor cells suggested a role for APRIL in
tumorigenesis. Similarly, the monitoring of APBF transcript or
polypeptide production, or gene expression level, or fluctuations
therein, in one or more tumor biopsy samples is expected to provide
information relevant to diagnosis, prognostication and/or staging
of neoplastic disease in a cancer sufferer.
[0057] Any suitable means for detecting APBF transcript or
polypeptide production or stabilization, or gene expression level,
can be applied for the present diagnostic purposes. Appropriate
methods are described in Sambrook et al. (1989), Molecular Cloning:
A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.
[0058] Standard methods of analysis allow the detection of activity
by cells in response to ligand binding. For example preparations of
the APBF heteromer will be useful in analyses of cellular
proliferation, differentiation, and apoptosis. Numerous cell types
can be rapidly screened in such a manner using standard methods
such as radioactive-thymidine incorporation, cell cycle analysis,
and MTT uptake and conversion (detailed in Celis et al., Cell
Biology, A Laboratory Handbook, Volume One, Academic Press, San
Diego, Calif. (1997). Other methods of analysis that can be used to
assess activity include protein phosphorylation analysis, for
example, of Nuclear Factor .kappa.B transcription factor
(NF.kappa.B) or c-Jun N-terminal Kinase (JNK) (eg., Mackay et al.,
J. Biol. Chem. 271: 24934-24938 (1996); Wong et al., J. Biol. Chem.
272: 25190-25194 (1997)). Other readily accessable assays include
measurements of cytokine secretion (eg. II-8: Chicheportiche et
al., J. Biol. Chem. 272: 32401-32410 (1997)), calcium flux, pH
change, cell/cell adhesion, etc (with references).
[0059] In addition to these readouts, analyses of upregulated or
downregulated genes are readily done, for, by example, Northern
blot, targeted array, or gene array analyses ( eg. Teague et al.,
Proc. Natl. Acad. Sci. USA 96 :12691-12696 (1999); Lockhart et al.,
Nat. Biotechnol. 14: 1675-1680 (1996)). Such differential gene
expression studies identify specific sets of genes which respond to
ligand activity, and can provide detailed profiles of ligand
function (eg. Jiang et al., Oncogene 11: 1179-1189 (1995)).
Particularly sensitive to such analyses are modifiers of cell
growth, eg. growth hormone receptor genes, transcription factors,
genes whose proteins induce or block cell death, and cell cycle
mediators, among many others.
[0060] The present invention is useful for diagnosis or treatment
of various immune system-related disorders in mammals, preferably
humans. Such disorders include but are not limited to cancer,
including, but not limited to, cellular disorders as, for example,
renal cell cancer, Kaposi's sarcoma, chronic leukemia, breast
cancer, sarcoma, ovarian carcinoma, rectal cancer, throat cancer,
melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer,
mammary adenocarcinoma, pharyngeal squamous cell carcinoma, and
gastrointestinal or stomach cancer. Additionally, the present
invention is useful for the treatment of proliferative conditions
that are not considered to be tumors, i.e. cellular
hyperproliferation (hyperplasia), such as, for example,
scleroderma, pannus formation in rheumatoid arthritis, postsurgical
scarring and lung, liver and uterine fibrosis. In addition, the
present invention is useful for the treatment of
immunodeficiencies, inflammatory diseases, lymphadenopathy,
autoimmune diseases, and graft versus host disease.
[0061] In one embodiment, conditions caused by a decrease in the
normal level of APBF activity in an individual can be treated by
administration of APBF or an agonist to APBF, where an agonist to
APBF refers to any natural or synthetic composition which
potentiates function, where function refers to any measurable
effect of APBF interaction with cells, tissues or organisms, as
measured in any known in vitro or in vivo assays, which is mediated
by APBF. In one embodiment, APBF is in soluble form. The invention
also provides a method of treatment of disorders caused by an
increase in the normal level of APBF activity in an individual by
administration of an antagonist to APBF, where an antagonist to
APBF refers to any natural or synthetic composition that blocks
function, where function refers to any measurable effect of APBF
interaction with cells, tissues or organisms, as measured in any
known in vitro or in vivo assays, which is mediated by APBF
heteromers.
[0062] Pharmaceutical compositions of the invention may comprise a
therapeutically effective amount of APBF, or its receptor, or
fragments or mimetics thereof, and, optionally may include
pharmaceutically acceptable carriers. Accordingly, this invention
provides methods for treatment of cancer, and methods of
stimulating, or in certain instances, inhibiting the immune system,
or parts thereof by administering a pharmaceutically effective
amount of a compound of the invention or its pharmaceutically
acceptable salts or derivatives. In certain preferred embodiments,
the invention relates to methods for inhibiting B-cell growth,
T-cell growth or tumor cell growth by administering a
therapeutically effective amount of an isolated APBF polypeptide or
active fragment thereof, or a recombinant APBF molecule or active
fragment thereof, or an antibody specific for APBF or active
fragment thereof. In the context of this invention "inhibition"
relates to any and all mechanisms for reducing or ameliorating
activity, including inducing cell death (apoptosis). It should of
course by understood that the compositions and methods of this
invention can be used in combination with other therapies for
various treatments.
[0063] The compositions can be formulated for a variety of routes
of administration, including systemic, topical or localized
administration. For systemic administration, injection is
preferred, including intramuscular, intravenous, intraperitoneal,
and subcutaneous for injection, the compositions of the invention
can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the compositions may be formulated
in solid form and, optionally, redissolved or suspended immediately
prior to use. Lyophilized forms are also included in the
invention.
[0064] The compositions can be administered orally, or by
transmucosal or transdermal means. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are known in
the art, and include, for example, for transmucosal administration,
bile salts, fusidic acid derivatives, and detergents. Transmucosal
administration may be through nasal sprays or using suppositories.
For oral administration, the compositions are formulated into
conventional oral administration forms such as capsules, tablets,
and tonics. For topical administration, the compositions of the
invention are formulated into ointments, salves, gels, or creams as
known in the art.
[0065] The dose and dosing regimen will depend on the type of
disease, the patient and the patient's history. In one embodiment
the disease is cancer. The amount must be effective to treat,
suppress, or alter the progression of the disease. The doses may be
single doses or multiple doses. If multiple doses are employed, as
preferred, the frequency of administration will depend, for
example, on the type of host and and type of disease, dosage
amounts etc. For some types of cancers or cancer lines, daily
administration will be effective, whereas for others,
administration every other day or every third day will be
effective. The amount of active compound administered at one time
or over the course of treatment will depend on many factors. For
example, the age and size of the subject, the severity and course
of the disease being treated, the manner and form of
administration, and the judgments of the treating physician.
However, an effective dose may be in the range of from about 0.005
to about 5 mg/kg/day, preferably about 0.05 to about 0.5 mg/kg/day.
The dosage amount which will be most effective will be one which
results in no tumor appearance or complete regression of the tumor,
and is not toxic to the patient. One skilled in the art will
recognize that lower and higher doses may also be useful.
EXAMPLES
Example 1
[0066] This example describes the detection of APRIL and BAFF
heteromers by immunoprecipitation following co-transfection into
mammalian cells.
[0067] Methods:
[0068] The plasmids encoding FLAG-tagged human soluble APRIL,
beginning with residue A105 (LT033) or K110 (PL448), soluble
FLAG-tagged human TWEAK beginning at A106 (PS288) or soluble
FLAG-tagged human EDA beginning at A242 (PS548) or empty vector
(CH269) were co-transfected with a full-length human BAFF construct
(PS544) into 293T cells using lipofectamine (Life Technologies). At
48 hrs. post-transfection, conditioned media was collected and used
for immunoprecipitation experiments. The immunoprecipitation
samples contained 200 .mu.l of conditioned media, 5 .mu.g/ml of the
anti-FLAG antibody M2 (Sigma) and 800 .mu.l of DMEM containing 10%
FCS, glutamine, Pen-Strep, G418 and sodium azide and were incubated
at 4.degree. C. for 1 hour, with agitation. Then, 30 .mu.l of
ProteinA-Sepharose beads (Pharmacia) was added to the samples and
the mixture was incubated overnight at 4.degree. C. with agitation.
The beads were collected by centrifugation and then washed one time
with the DMEM media described above and then 3 times with PBS. The
final pellet containing the beads was then suspended in 2.times.SDS
non-reducing sample buffer and boiled for 5 minutes. The beads were
spun out and 25 .mu.l of the supernatant was loaded onto 2 separate
4-20% SDS-PAGE gradient gels (Novex). In order to examine the level
of ligand expression, non-immunoprecipitated conditioned media from
the co-transfected cells were also loaded. These samples were
diluted two fold with 2.times.non-reducing sample buffer, boiled
for 2 min. and then 25 .mu.l was loaded into each lane. Each gel
contained a set of immunoprecipitations and a set of
non-immunoprecipitated conditioned media. After the gels were
transferred to Immobilon (Millipore) filters using a BioRad
apparatus, the filters were blocked in 5% non-fat dry milk diluted
in TBST for 1 hr at room temperature. The filters were then
separated and one was incubated with 5 .mu.g/ml of the biotinylated
anti-FLAG antibody, M2 and the other was incubated with 1 .mu.g/ml
of an anti-human BAFF antibody 53.14 (rat IgM) for about 1 hr at
room temperature. The filters were washed with 3 changes of TBST
and then incubated in a 1:3000 dilution of streptavidin-HRP or
anti-rat IgM-HRP (Jackson ImmunoResearach) for 30 min. at room
temperature. The filters were again washed 3 times and then
detected using ECL reagents (Amersham). The filters were exposed to
x-ray film for various lengths of time.
[0069] The results of the co-expression experiment are shown in
FIG. 3. Panel A, lanes 1-5, show western blots of straight
conditioned media from cells co-transfected with various human
soluble TNF family ligands and human full length BAFF encoding
plasmids. Panel A, lanes 7-12, show western blots of the straight
supernatant after immunoprecipitation with an anti-FLAG antibody.
The detection reagent used in Panel A is an anti-FLAG antibody.
Lane 1: FLAG-tagged human soluble APRIL A105 (beginning at residue
A105)+human full length BAFF; Lane 2: FLAG-tagged human soluble
APRIL K110 (beginning at residue K110)+human full length BAFF; Lane
3: FLAG-tagged human soluble TWEAK+human full length BAFF; Lane 4:
FLAG-tagged human EDA+human full length BAFF; Lane 5: empty control
vector; Lane 6: molecular weight standards (Benchmark,
LifeTechnologies) in kDa, 185, 119, 85, 62, 51, 38.2, 26.0, 20.2,
14.5, 9.1; Lane 7-11 correspond to Lanes 1-5, respectively, after
immunoprecipitation with an anti-FLAG antibody; Lane 12: purified
human FLAG-BAFF Q136, 5 ng.
[0070] Panel B, lanes 1-5, show western blots of straight
supernatant from cells co-transfected with various APRIL and BAFF
encoding plasmids. The detection reagent is an anti-BAFF antibody.
Panel B, lanes 7-11 show western blots of immunoprecipitates in
which cells were co-transfected with various APRIL and BAFF
encoding plasmids and immunoprecipitated with an anti-FLAG
antibody. The detection reagent is an anti-BAFF antibody. Lane 1:
FLAG-tagged human soluble APRIL A105 (beginning at residue
A105)+human full length BAFF; Lane 2: FLAG-tagged human soluble
APRIL K110 (beginning at residue K110)+human full length BAFF; Lane
3: FLAG-tagged human soluble TWEAK A106+human full length BAFF;
Lane 4: FLAG-tagged human EDA A242+human full length BAFF; Lane 5:
empty control vector; Lane 6: molecular weight standards
(Benchmark, LifeTechnologies) in kDa, 185, 119, 85, 62, 51, 38.2,
26.0, 20.2, 14.5, 9.1; Lane 7-11 correspond to Lanes 1-5,
respectively, after immunoprecipitation with an anti-FLAG antibody;
Lane 12: purified human FLAG-BAFF Q136, 5 ng.
[0071] In panel A, the detection with the anti-FLAG antibody, M2
shows that all the FLAG-epitope tagged soluble ligands are
expressed and secreted into the cell culture of the transfected
cells. The two APRIL constructs shown in lanes 1 and 2 are
expressed about 5 fold lower than TWEAK (lane 3) or EDA (lane 4).
No protein is visible in lane 5, which is the control vector lane.
Lanes 7-11 represent the FLAG-tagged proteins after they are
immunoprecipitated. Here both APRIL proteins (lanes 7 and 8), TWEAK
(lane 9) and EDA (lane 10) are precipitated and detected by M2.
Lane 12 is approximately 5 ng of FLAG-BAFF for a standard.
[0072] In panel B, lanes 1-5 represent the detection of BAFF in the
co-transfected cell culture media. BAFF is expressed in combination
with all the FLAG-epitope tagged soluble ligands and is slightly
higher in the APRIL co-transfections (lanes 1 and 2). On the right
side of the blot in panel B are the M2 immunoprecipitations
detected with the anti-BAFF antibody, 53.14. Here, BAFF is only
immunoprecipitated in combination with APRIL (lanes 7 and 8) and
not TWEAK or EDA. The standard in lane 12 indicates the size of a
soluble FLAG-BAFF molecule expressed in 293T cells, which is
approximately the molecular weight of the naturally cleaved
molecule. This demonstrates that BAFF and APRIL form a heteromeric
complex. The co-immunoprecipitations have also been evaluated in
the presence of 1 M NaCl and the results are the same.
Example 2
[0073] This example describes the detection of APBF heteromers by
immunoprecipitation following co-transfection of two soluble
constructs into mammalian cells.
[0074] Methods:
[0075] Plasmids encoding the following human soluble TNF family
ligands were constructed with the indicated N-terminal epitope tags
beginning at the ligand amino acid residue indicated in a PCR3
based mammalian cell expression vector: FLAG-APRIL, beginning with
residue A105 (plasmid #LT033) or H115 (plasmid #LT038), FLAG-TWEAK
A106 (plasmid #PS288), myc-APRIL A105 (plasmid #JST557), and
myc-BAFF Q136 (plasmid #JST556). Various constructs encoding
FLAG-tagged ligands, full length murine APRIL(plasmid #LT022), or
empty vector control (plasmid #CH269) were each co-transfected with
the myc-BAFF Q136 construct into 293T cells using lipofectamine
(Life Technologies, Gaithersburg, Md.). At 48 hrs.
post-transfection, conditioned media was collected and used for
immunoprecipitation experiments. The immunoprecipitation samples
contained 100 .mu.l of conditioned media, 5 .mu.g/ml of the
anti-FLAG antibody M2 (Sigma, St Louis, Mo.) and 900 .mu.l of DMEM
containing 10% FCS, glutamine, Pen-Strep, G418 and sodium azide and
were incubated at 4.degree. C. for 1 hour, with agitation. Then, 30
.mu.l of ProteinA-Sepharose beads (Amersham Pharmacia, Piscataway,
N.J.) was added to the samples and the mixture was incubated
overnight at 4.degree. C. with agitation. The beads were collected
by centrifugation and then washed one time with the DMEM media
described above and then 3 times with PBS. The final pellet
containing the beads was then suspended in 2.times.SDS non-reducing
sample buffer and boiled for 5 minutes. The beads were spun out and
25 .mu.l of the supernatant was loaded onto a 4-20% SDS-PAGE
gradient gel (Novex, San Diego, Calif.). After the gel was
transferred to Immobilon (Millipore, Bedford, Mass.) using a BioRad
apparatus, the filters were blocked in 5% non-fat dry milk diluted
in TBST for 1 hr at room temperature. The filter was then incubated
with 1 .mu.g/ml of anti-myc antibody 9E10. The filter was washed
with 3 changes of TBST and then incubated in a 1:3000 dilution of
anti-mouse IgG-HRP (Jackson ImmunoResearch, West Grove, Pa.) for 30
min. at room temperature. The filter was again washed 3 times and
then detected using ECL reagents (Amersham Pharmacia, Piscataway,
N.J.). The filter was exposed to x-ray film for various lengths of
time.
[0076] The results shown in FIG. 4 show a western blot of the
immunoprecipiations of conditioned media from cells co-transfected
with plasmids encoding various soluble APRIL and soluble BAFF
proteins and immunoprecipitated with an anti-FLAG-tagged antibody.
The detection reagent for the western blot is an anti-myc antibody,
9E10.
[0077] Lanes 1-6, show western blots of straight conditioned media
from cells co-transfected with plasmids encoding various human
soluble TNF family ligands and human soluble myc-BAFF Q136. Lane 7
is a molecular weight marker. Lanes 8-12, show western blots of the
conditioned media after immunoprecipitation with an anti-FLAG
antibody. The detection reagent used an anti-MYC antibody, 9E10.
Lane 1: FLAG-tagged human soluble TWEAK A106+human soluble myc-BAFF
Q136; Lane 2: FLAG-tagged human soluble APRIL HI 15+human soluble
myc-BAFF Q136; Lane 3: MYC-tagged human soluble APRIL A105+human
soluble myc-BAFF Q136; Lane 4: FLAG-tagged human soluble APRIL
A105+human soluble myc-BAFF Q136; Lane 5: full length murine
APRIL+human soluble myc-BAFF Q136; Lane 6: empty vector
control+human soluble myc-BAFF Q136; Lane 7, molecular weight
standards (Benchmark, LifeTechnologies) in kDa, 38.2, 26.0, 20.2,
14.5; Lanes 8-12 correspond to Lanes 1-5, respectively, after
immunoprecipitation with an anti-FLAG antibody.
[0078] In lanes 1-6, the western blot of the conditioned media as
detected by anti-MYC antibody 9E10 shows that all co-transfected
293T cells express and secrete myc-Baff Q136 into the cell culture
media in nearly equal amounts except for the FLAG-TWEAK+myc-Baff
Q136 (lane 1) which shows significantly lower amounts of myc-BAFF.
Lanes 8-12 show immunoprecipitation of conditioned media with
anti-FLAG antibody followed by detection on western blot with
anti-myc antibody 9E10. Lanes 10 and 12 show conditioned media of
myc-BAFF co-transfected with myc-APRIL A105 or full length murine
APRIL, respectively, and serve as negative controls in that neither
APRIL construct contains the flag epitope and therefore were not
immunoprecipitated by the anti-flag antibody. The FLAG-TWEAK
co-transfection shows no band corresponding to myc-BAFF Q136, even
upon overexposure, and therefore does not interact with MYC-BAFF.
Only lanes 9 and 11, those containing myc-Baff Q136 co-expressed
with FLAG-APRIL molecules H115 and A105 respectively, show the
myc-baff band after anti-FLAG immunoprecipitation. Bands of
approximately 18 kDa, the predicted size of myc-BAFF Q136, are
observed in each lane. The intensity of the band co-expressed with
FLAG-APRIL A87 (lane 3) greater than that co-expressed with
FLAG-APRIL H97 (lane 4). This indicates that only soluble
FLAG-APRIL ligands were able to interact with soluble MYC-BAFF to
form heteromeric complexes.
[0079] This demonstrates that soluble forms of BAFF and APRIL have
the ability to form a heteromeric complex, and that no cell
associated form appears to be required for heteromer formation.
Example 3
Production and isolation of APBF by Affinity Methods
[0080] Plasmids encoding the following human soluble TNF family
ligands were constructed with N-terminal FLAG or 6.times.His
epitope tags beginning at the amino acid residue indicated in a
PCR3 based mammalian cell expression vector: FLAG-APRIL, beginning
with residue A87 (plasmid Lf133) and RGS(H)6-BAFF Q134. These
plasmids are then co-transfected into 293T cells using
lipofectamine (Life Technologies, Gaithersburg, Md.) and at 48 hrs.
post-transfection, conditioned media is collected. The conditioned
media is dialyzed against 50 mM NaH2PO4, pH8.0; 300 mM NaCl; 10 mM
imidazole and run over a Ni--NTA Superflow column (Qiagen,
Valencia, Calif.). Homomers and heteromers containing the
6.times.His tagged BAFF subunit bind to the Ni column; homomeric
FLAG-APRIL molecules flow through. The column is washed with 50 mM
NaH2PO4, pH8.0; 300 mM NaCl; 20 mM imidazole with 5-10 column
volumes. The column is eluted with 5 column volumes with 50 mM
NaH2PO4, pH8.0; 300 mM NaCl; 250mM imidazole. The eluted material
includes RGS(H)6-BAFF Q134 homomers and heteromers with FLAG-APRIL.
This eluted material is applied to an M1 or M2 anti-FLAG Ab
affinity column (Sigma, St. Louis, Mo.). A buffer exchange to 150
mM NaCl--50 mM Tris pH7.0 is performed, and the buffer adjusted to
2mM CaCl2 if using the M1 column (this Ab requires Ca for binding).
The column is washed with 150 mM NaCl--50 mM Tris pH7.0 (with 2mM
CaCl2 for M1). The M1 column is eluted by incubating the column
with 150 mM NaCl--50 mM Tris pH7.0--2 mM EDTA for 30 minutes,
followed by aliquots of 150 mM NaCl--50 mM Tris pH7.0-2 mM EDTA, 10
min incubations, 6 times. Alternatively, both the M1 and M2 columns
can be eluted by competition with FLAG peptide by allowing the
column to drain completely and eluting 5 times with one column
volume each of to 150 mM NaCl--50 mM Tris pH7.0 containing 100
.upsilon.g/ml FLAG peptide. Eluted material will contain only
native FLAG-APRIL:: RGS(H)6-BAFF Q134 heteromers.
[0081] Alternatively, cell lines or cells transfected, as above,
with plasmids encoding full length or untagged soluble APRIL and
BAFF constructs could be used as a source to isolate APBF complexes
with anti-peptide antibodies raised against regions of the
extracellular domains for APRIL and BAFF. These antibodies could be
coupled to a resin by conventional means. Conditioned media or cell
extracts of such cells could be run first over a column containing
the coupled antibody (s) against one of the ligands, for example
anti-BAFF antibodiy (s). In this instance, only homomers and
heteromers containing a BAFF subunit bind to the anti-BAFF column;
homomeric APRIL molecules flow through. After washing the column in
150 mM NaCl--50 mM Tris pH7.0, the bound molecules could be eluted
off by competition with the same BAFF peptide(s) used to raise the
anti-BAFF antibody(s) by allowing the column to drain completely
and eluting 5 times with one column volume each of to 150 mM
NaCl--50 mM Tris pH 7.0 containing 100 .mu.g/ml or greater the
peptide(s). This eluate could be dialyzed to remove the peptide(s)
and then similarly run over a column containing anti-peptide
antibody(s) raised against the other ligand, in this example APRIL.
). In this instance, homomeric BAFF molecules would not bind to the
column and flow through. Only the remaining APBF heteromers bind to
the anti-BAFF column. These APBF heteromers could be similarly
eluted by competition with the same APRIL peptide(s) used to
generate the anti-APRIL antibody(s).
Sequence CWU 1
1
8 1 1346 DNA Homo sapien 1 ggtacgaggc ttcctagagg gactggaacc
taattctcct gaggctgagg gagggtggag 60 ggtctcaagg caacgctggc
cccacgacgg agtgccagga gcactaacag tacccttagc 120 ttgctttcct
cctccctcct ttttattttc aagttccttt ttatttctcc ttgcgtaaca 180
accttcttcc cttctgcacc actgcccgta cccttacccg ccccgccacc tccttgctac
240 cccactcttg aaaccacagc tgttggcagg gtccccagct catgccagcc
tcatctcctt 300 tcttgctagc ccccaaaggg cctccaggca acatgggggg
cccagtcaga gagccggcac 360 tctcagttgc cctctggttg agttgggggg
cagctctggg ggccgtggct tgtgccatgg 420 ctctgctgac ccaacaaaca
gagctgcaga gcctcaggag agaggtgagc cggctgcagg 480 ggacaggagg
ccctcccaga atggggaagg gtatccctgg cagagtctcc cggagcagag 540
ttccgatgcc ctggaagcct gggagaatgg ggagagatcc cggaaaaggg agcagtgctc
600 acccaaaaac agaagaagca gcactctgtc ctgcacctgg ttcccattaa
cgccacctcc 660 aaggatgact ccgatgtgac agaggtgatg tggcaaccag
ctcttaggcg tgggagaggc 720 ctacaggccc aaggatatgg tgtccgaatc
caggatgctg gagtttatct gctgtatagc 780 caggtcctgt ttcaagacgt
gactttcacc atgggtcagg tggtgtctcg agaaggccaa 840 ggaaggcagg
agactctatt ccgatgtata agaagtatgc cctcccaccc ggaccgggcc 900
tacaacagct gctatagcgc aggtgtcttc catttacacc aaggggatat tctgagtgtc
960 ataattcccc gggcaagggc gaaacttaac ctctctccac atggaacctt
cctggggttt 1020 gtgaaactgt gattgtgtta taaaaagtgg ctcccagctt
ggaagaccag ggtgggtaca 1080 tactggagac agccaagagc tgagtatata
aaggagaggg aatgtgcagg aacagaggca 1140 tcttcctggg tttggctccc
cgttcctcac ttttcccttt tcattcccac cccctagact 1200 ttgattttac
ggatatcttg cttctgttcc ccatggagct ccgaattctt gcgtgtgtgt 1260
gatgagggg cgggggacgg gcgccaggca ttgttcagac ctggtcgggg cccactggaa
1320 catccagaa cagcaccacc atctta 1346 2 250 PRT Homo sapien 2 Met
Pro Ala Ser Ser Pro Phe Leu Leu Ala Pro Lys Gly Pro Pro Gly 1 5 10
15 Asn Met Gly Gly Pro Val Arg Glu Pro Ala Leu Ser Val Ala Leu Trp
20 25 30 Leu Ser Trp Gly Ala Ala Leu Gly Ala Val Ala Cys Ala Met
Ala Leu 35 40 45 Leu Thr Gln Gln Thr Glu Leu Gln Ser Leu Arg Arg
Glu Val Ser Arg 50 55 60 Leu Gln Gly Thr Gly Gly Pro Ser Gln Asn
Gly Glu Gly Tyr Pro Trp 65 70 75 80 Gln Ser Leu Pro Glu Gln Ser Ser
Asp Ala Leu Glu Ala Trp Glu Asn 85 90 95 Gly Glu Arg Ser Arg Lys
Arg Arg Ala Val Leu Thr Gln Lys Gln Lys 100 105 110 Lys Gln His Ser
Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys 115 120 125 Asp Asp
Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg 130 135 140
Gly Arg Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala 145
150 155 160 Gly Val Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val
Asp Phe 165 170 175 Thr Met Gly Gln Val Val Ser Arg Glu Gly Gln Gly
Arg Gln Glu Thr 180 185 190 Leu Phe Arg Cys Ile Arg Ser Met Pro Ser
His Pro Asp Arg Ala Tyr 195 200 205 Asn Ser Cys Tyr Ser Ala Gly Val
Phe His Leu His Gln Gly Asp Ile 210 215 220 Leu Ser Val Ile Ile Pro
Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro 225 230 235 240 His Gly Thr
Phe Leu Gly Phe Val Lys Leu 245 250 3 916 DNA Homo sapien 3
gaattcggca gcaggctcca ggccacatgg ggggctcagt cagagagcca gccctttcgg
60 ttgctctttg gttgagttgg ggggcagttc tgggggctgt gacttgtgct
gtcgcactac 120 tgatccaaca gacagagctg caaagcctaa ggcgggaggt
gagccggctg cagcggagtg 180 gagggccttc ccagaagcag ggagagcgcc
catggcagag cctctgggag cagagtcctg 240 atgtcctgga agcctggaag
gatggggcga aatctcggag aaggagagca gtactcaccc 300 agaagcacaa
gaagaagcac tcagtcctgc atcttgttcc agttaacatt acctccaagg 360
actctgacgt gacagaggtg atgtggcaac cagtacttag gcgtgggaga ggccctggag
420 gcccagggag acattgtacg agtctgggac actggaattt atctgctcta
tagtcaggtc 480 ctgtttcatg atgtgacttt cacaatgggt caggtggtat
ctcgggaagg acaagggaga 540 agagaaactc tattcgatgt atcagaagta
tgccttctga tcctgaccgt gcctacaata 600 gctgctacag tgcaggtgtc
tttcatttac atcaagggga tattatcact gtcaaaattc 660 cacgggcaaa
cgcaaaactt agcctttctc cgcatggaac attcctgggg tttgtgaaac 720
tatgattgtt ataaaggggg tggggatttc ccattccaaa aactggctag acaaaggaca
780 aggaacggtc aagaacagct ctccatggct ttgccttgac tgttgttcct
ccctttgcct 840 ttcccgctcc cactatctgg gctttgactc catggatatt
aaaaaagtag aatattttgt 900 gtttatctcc caaaaa 916 4 232 PRT Homo
sapien 4 Met Gly Gly Ser Val Arg Glu Pro Ala Leu Ser Val Ala Leu
Trp Leu 1 5 10 15 Ser Trp Gly Ala Val Leu Gly Ala Val Thr Cys Ala
Val Ala Leu Leu 20 25 30 Ile Gln Gln Thr Glu Leu Gln Ser Leu Arg
Arg Glu Val Ser Arg Leu 35 40 45 Gln Arg Ser Gly Gly Pro Ser Gln
Lys Gln Gly Glu Arg Pro Trp Gln 50 55 60 Ser Leu Trp Glu Gln Ser
Pro Asp Val Leu Glu Ala Trp Lys Asp Gly 65 70 75 80 Ala Lys Ser Arg
Arg Arg Arg Ala Val Leu Thr Gln Lys His Lys Lys 85 90 95 Lys His
Ser Val Leu His Leu Val Pro Val Asn Ile Thr Ser Lys Asp 100 105 110
Ser Asp Val Thr Glu Val Met Trp Gln Pro Val Leu Arg Arg Gly Arg 115
120 125 Gly Pro Gly Gly Gln Gly Asp Ile Val Arg Val Trp Asp Thr Gly
Ile 130 135 140 Tyr Leu Leu Tyr Ser Gln Val Leu Phe His Asp Val Thr
Phe Thr Met 145 150 155 160 Gly Gln Val Val Ser Arg Glu Gly Gln Gly
Arg Arg Glu Thr Leu Phe 165 170 175 Arg Cys Ile Arg Ser Met Pro Ser
Asp Pro Asp Arg Ala Tyr Asn Ser 180 185 190 Cys Tyr Ser Ala Gly Val
Phe His Leu His Gln Gly Asp Ile Ile Thr 195 200 205 Val Lys Ile Pro
Arg Ala Asn Ala Lys Leu Ser Leu Ser Pro His Gly 210 215 220 Thr Phe
Leu Gly Phe Val Lys Leu 225 230 5 1052 DNA Homo sapien 5 tgccaagccc
tgccatgtag tgcacgcagg acatcaacaa acacagataa caggaaatga 60
tccattccct gtggtcactt attctaaagg ccccaacctt caaagttcaa gtagtgatat
120 ggatgactcc acagaaaggg agcagtcacg ccttacttct tgccttaaga
aaagagaaga 180 aatgaaactg aaggagtgtg tttccatcct cccacggaag
gaaagcccct ctgtccgatc 240 ctccaaagac ggaaagctgc tggctgcaac
cttgctgctg gcactgctgt cttgctgcct 300 cacggtggtg tctttctacc
aggtggccgc cctgcaaggg gacctggcca gcctccgggc 360 agagctgcag
ggccaccacg cggagaagct gccagcagga gcaggagccc ccaaggccgg 420
cctggaggaa gctccagctg tcaccgcggg actgaaaatc tttgaaccac cagctccagg
480 agaaggcaac tccagtcaga acagcagaaa taagcgtgcc gttcagggtc
cagaagaaac 540 agtcactcaa gactgcttgc aactgattgc agacagtgaa
acaccaacta tacaaaaagg 600 atcttacaca tttgttccat ggcttctcag
ctttaaaagg ggaagtgccc tagaagaaaa 660 agagaataaa atattggtca
aagaaactgg ttactttttt atatatggtc aggttttata 720 tactgataag
acctacgcca tgggacatct aattcagagg aagaaggtcc atgtctttgg 780
ggatgaattg agtctggtga ctttgtttcg atgtattcaa aatatgcctg aaacactacc
840 caataattcc tgctattcag ctggcattgc aaaactggaa gaaggagatg
aactccaact 900 tgcaatacca agagaaaatg cacaaatatc actggatgga
gatgtcacat tttttggtgc 960 attgaaactg ctgtgaccta cttacaccat
gtctgtagct attttcctcc ctttctctgt 1020 acctctaaga agaaagaatc
taactgaaaa ta 1052 6 285 PRT Homo sapien 6 Met Asp Asp Ser Thr Glu
Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu
Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys
Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45
Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50
55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu
Arg 65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala
Gly Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala
Val Thr Ala Gly Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly
Glu Gly Asn Ser Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val
Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135 140 Asp Cys Leu Gln Leu
Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys 145 150 155 160 Gly Ser
Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser 165 170 175
Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr 180
185 190 Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala
Met 195 200 205 Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly
Asp Glu Leu 210 215 220 Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn
Met Pro Glu Thr Leu 225 230 235 240 Pro Asn Asn Ser Cys Tyr Ser Ala
Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255 Asp Glu Leu Gln Leu Ala
Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270 Asp Gly Asp Val
Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280 285 7 1478 DNA Homo
sapien 7 gtggtcactt actccaaagg cctagacctt caaagtgctc ctcgtggaat
ggatgagtct 60 gcaaagaccc tgccaccacc gtgcctctgt ttttgctccg
agaaaggaga agatatgaaa 120 gtgggatatg atcccatcac tccgcagaag
gaggagggtg cctggtttgg gatctgcagg 180 gatggaaggc tgctggctgc
taccctcctg ctggccctgt tgtccagcag tttcacagcg 240 atgtccttgt
accagttggc tgccttgcaa gcagacctga tgaacctgcg catggagctg 300
cagagctacc gaggttcagc aacaccagcc gccgcgggtg ctccagagtt gaccgctgga
360 gtcaaactcc tgacaccggc agctcctcga ccccacaact ccagccgcgg
ccacaggaac 420 agacgcgctt tccagggacc agaggaaaca gaacaagatg
tagacctctc agctcctcct 480 gcaccatgcc tgcctggatg ccgccattct
caacatgatg ataatggaat gaacctcaga 540 aacagaactt acacatttgt
tccatggctt ctcagcttta aaagaggaaa tgccttggag 600 gagaaagaga
acaaaatagt ggtgaggcaa acaggctatt tcttcatcta cagccaggtt 660
ctatacacgg accccatctt tgctatgggt catgtcatcc agaggaagaa agtacacgtc
720 tttggggacg agctgagcct ggtgaccctg ttccgatgta ttcagaatat
gcccaaaaca 780 ctgcccaaca attcctgcta ctcggctggc atcgcgaggc
tggaagaagg agatgagatt 840 cagcttgcaa ttcctcggga gaatgcacag
atttcacgca acggagacga caccttcttt 900 ggtgccctaa aactgctgta
actcacttgc tggagtgcgt gatccccttc cctcgtcttc 960 tctgtacctc
cgagggagaa acagacgact ggaaaaacta aaagatgggg aaagccgtca 1020
gcgaaagttt tctcgtgacc cgttgaatct gatccaaacc aggaaatata acagacagcc
1080 acaaccgaag tgtgccatgt gagttatgag aaacggagcc cgcgctcaga
aagaccggat 1140 gaggaagacc gttttctcca gtcctttgcc aacacgcacc
gcaaccttgc tttttgcctt 1200 gggtgacaca tgttcagaat gcagggagat
ttccttgttt tgcgatttgc catgagaaga 1260 gggcccacaa ctgcaggtca
ctgaagcatt cacgctaagt ctcaggattt actctccctt 1320 ctcatgctaa
gtacacacac gctcttttcc aggtaactac tatgggatac tatggaaagg 1380
ttgtttgttt ttaaatctag aagtcttgaa ctggcaatag acaaaaatcc ttataaattc
1440 aagtgtaaaa taaacttaat taaaaaggtt taagtgtg 1478 8 290 PRT Homo
sapien 8 Met Asp Glu Ser Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys
Phe Cys 1 5 10 15 Ser Glu Lys Gly Glu Asp Met Lys Val Gly Tyr Asp
Pro Ile Thr Pro 20 25 30 Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile
Cys Arg Asp Gly Arg Leu 35 40 45 Leu Ala Ala Thr Leu Leu Leu Ala
Leu Leu Ser Ser Ser Phe Thr Ala 50 55 60 Met Ser Leu Tyr Gln Leu
Ala Ala Leu Gln Ala Asp Leu Met Asn Leu 65 70 75 80 Arg Met Glu Leu
Gln Ser Tyr Arg Gly Ser Ala Thr Pro Ala Ala Ala 85 90 95 Gly Ala
Pro Glu Leu Thr Ala Gly Val Lys Leu Leu Thr Pro Ala Ala 100 105 110
Pro Arg Pro His Asn Ser Ser Arg Gly His Arg Asn Arg Arg Ala Phe 115
120 125 Gln Gly Pro Glu Glu Thr Glu Gln Asp Val Asp Leu Ser Ala Pro
Pro 130 135 140 Ala Pro Cys Leu Pro Gly Cys Arg His Ser Gln His Asp
Asp Asn Gly 145 150 155 160 Met Asn Leu Arg Asn Arg Thr Tyr Thr Phe
Val Pro Trp Leu Leu Ser 165 170 175 Phe Lys Arg Gly Asn Ala Leu Glu
Glu Lys Glu Asn Lys Ile Val Val 180 185 190 Arg Gln Thr Gly Tyr Phe
Phe Ile Tyr Ser Gln Val Leu Tyr Thr Asp 195 200 205 Pro Ile Phe Ala
Met Gly His Val Ile Gln Arg Lys Lys Val His Val 210 215 220 Phe Gly
Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn 225 230 235
240 Met Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala
245 250 255 Arg Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro Arg
Glu Asn 260 265 270 Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe Phe
Gly Ala Leu Lys 275 280 285 Leu Leu 290
* * * * *