THIS APPLICATION HAS BEEN DIVIDED OUT OF INDIAN
APPLICATION NO. 864/KOLNP/2005
FIELD OF THE INVENTION
The invention relates generally to nucleic acids and polypeptides and more specifically to
nucleic acids and polypeptides encoding polypeptides useful for detecting and treating; lupus
nephritis, as well as for identifying therapeutic agents for treating the same.
BACKGROUND OF THE INVENTION
Lupus nephritis is an example of a "classical" auto-immune disease in which the
patient's immune system attacks his/her own organs. It has been estimated that 45-75% of lupus
patients eventually suffer from some form or other of kidney damage. Lupus varies greatly in
severity from mild cases requiring minimal intervention to those in which significant damage
occurs to vital organs such as lungs, kidneys, heart and brain, and which ultimately can be fetal.
Lupus is predominantly a female disease, with an approximate female to male ratio being 9:1. In
North America, it is estimated to affect 1 in 500 females mainly between the age of 20 to 40
years.
There is no known cure for lupus. Treatment is typically directed at controlling the
symptoms with the hope of putting the disease into remission. Recently, the antibiotic
rapamycin has been demonstrated to be an effective therapy in treating lupus nephritis in a
murine model of the disease.
1A

SUMMARY OF THE INVENTION
The invention is based, in part, upon the discovery of a gene, named BFLPC169, whose
expression is increased in kidney tissue in mice with lupus nephritis; however, the expression
level of the gene does not decrease markedly in response to treatment with rapamycin. This
expression profile indicates that the product of the BFLP0169 gene interacts with rapamycin
when this antibiotic is administered to ameliorate the symptoms of lupus nephritis. In the
absence of rapamycin, the gene product is free to bring about the diseased state, and its effects
can include the activation of genes required to bring about the diseased state. In the presence of
rapamycin, the BFLP0169 gene product is inactive and the diseased state diminishes.
Accordingly, the BFLP0169 protein is useful as a target for identifying agents that, like
rapamycin, are useful in treating symptoms of lupus nephritis.
In one aspect, the invention provides an isolated nucleic acid molecule that includes the
sequence of a nucleotide sequence encoding a BFLP0169 gene product. In a preferred
embodiment, the nucleotide sequence includes the sequence of SEQ ID NO: 1, or a fragment,
homolog, analog or derivative thereof. The nucleic acid can include, e.g., a nucleic acid
sequence encoding a polypeptide at least 70%, e.g., 80%, 85%, 90%, 95%, 98%, or even 99% or
more identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:2. The
nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
Also included in the invention is a vector containing one or more of the nucleic acids
described herein, and a cell containing the vectors or nucleic acids described herein.
The invention is also directed to host cells transformed with a vector comprising any of
the nucleic acid molecules described above.
In another aspect, the invention includes a pharmaceutical composition that includes a
BFLP0169 nucleic acid and a pharmaceutically acceptable carrier or diluent.
In a further aspect, the invention includes a substantially purified BFLP0169 polypeptide,
e.g., any of the BFLP0169 polypeptides encoded by a BFLP0169 nucleic acid, and fragments,
homologs, analogs, and derivatives thereof. The invention also includes a pharmaceutical
2

composition that includes a BFLP0169 polypeptide and a pharmaceutically acceptable carrier or
diluent.
In a still further aspect, the invention provides an antibody that binds specifically to a
BFLP0169 polypeptide. The antibody can be, e.g., a monoclonal or polyclonal antibody, and
fragments, homologs, analogs, and derivatives thereof. The invention also includes a
pharmaceutical composition including BFLP0169 antibody and a pharmaceutically acceptable
carrier or diluent. The invention is also directed to isolated antibodies that bind to an epitope on
a polypeptide encoded by any of the nucleic acid molecules described above.
The invention also includes kits comprising in one or more containers one or more of a
compound that is a BFLP0169 nucleic acid, a BFLP0169 polypeptide and/or an antibody to a
BFLP0169 polypeptide. The kit is preferably provided with instructions for use. If desired, the
compounds in the kits are provided along with a pharmaceutically acceptable carrier.
The invention further provides a method for producing a BFLP0169 polypeptide by
providing a cell containing a BFLP0169 nucleic acid, e.g., a vector that includes a BFLP0169
nucleic acid, and culturing the cell under conditions sufficient to express the BFLPO 169
polypeptide encoded by the nucleic acid. The expressed BFLPO 169 polypeptide is then
recovered from the cell. Preferably, the cell produces little or no endogenous BFLPO 169
polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
The invention is also directed to methods of identifying a BFLPO 169 polypeptide or
nucleic acid in a sample by contacting the sample with a compound that specifically binds to the
polypeptide or nucleic acid, and detecting complex formation, if present.
The invention further provides methods of identifying a compound that modulates the
activity of a BFLP0169 polypeptide by contacting a BFLP0169 polypeptide with a compound
and determining whether the BFLPO 169 polypeptide activity is modified.
The invention is also directed to compounds that modulate BFLPO 169 polypeptide
activity identified by contacting a BFLPO 169 polypeptide with the compound and determining
whether the compound modifies activity of the BFLPO 169 polypeptide, binds to the BFLPO 169
polypeptide, or binds to a nucleic acid molecule encoding a BFLPO 169 polypeptide.
3

In another aspect, the invention provides a method of determining the presence of or
predisposition of a BFLP0169 -associated disorder in a subject. The method includes providing a
sample from the subject and measuring the amount of BFLP0169 polypeptide in the subject
sample. The amount of BFLP0169 polypeptide in the subject sample is then compared to the
amount of BFLP0169 polypeptide in a control sample. An alteration in the amount of BFLP0169
polypeptide in the subject protein sample relative to the amount of BFLP0169 polypeptide in the
control protein sample indicates the subject has a tissue proliferation-associated condition. A
control sample is preferably taken from a matched individual, i.e., an individual of similar age,
sex, or other general condition but who is not suspected of having a tissue proliferation-
associated condition. Alternatively, the control sample may be taken from the subject at a time
when the subject is not suspected of having a tissue proliferation-associated disorder. In some
embodiments, the BFLP0169 is detected using a BFLP0169 antibody.
In a further aspect, the invention provides a method of determining the presence of or
predisposition of a BFLP0169 -associated disorder in a subject. The method includes providing a
nucleic acid sample, e.g., RNA or DNA, or both, from the subject and measuring the amount of
the BFLP0169 nucleic acid in the subject nucleic acid sample. The amount of BFLP0169 nucleic
acid sample in the subject nucleic acid sample is then compared to the amount of a BFLP0169
nucleic acid in a control sample. An alteration in the amount of BFLP0169 nucleic; acid in the
sample relative to the amount of BFLP0169 in the control sample indicates the subject has a
tissue proliferation-associated disorder.
In a still further aspect, the invention provides a method of treating or preventing or
delaying a BFLP0169 -associated disorder. The method includes administering to a subject in
which such treatment or prevention or delay is desired a BFLP0169 nucleic acid, a BFLP0169
polypeptide, or a BFLPO 169 antibody in an amount sufficient to treat, prevent, or delay a tissue
proliferation-associated disorder in the subject. Examples of such disorders include rheumatoid
arthritis and multiple sclerosis.
Unless otherwise defined, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. Although methods and materials similar or equivalent to those described herein can be
4

used in the practice or testing of the present invention, suitable methods and materials are
described below. All publications, patent applications, patents, and other references mentioned
herein are incorporated by reference in their entirety. In the case of conflict, the present
specification, including definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a histogram showing relative levels of gene expression in the mouse ortholog of
the human BFLP0169 gene in NZB x NZWF1 kidneys before, during, and after rapamycin
treatment, as well as in various control mouse strains and conditions.
DETAILED DESCRIPTION OF THE INVENTION
The BFLP0169 nucleic acid sequences disclosed herein were identified based on changes
in expression of the gene in kidneys of a lupus nephritis model mouse as compared to expression
of the gene in kidneys from non non-diseased mice. More particularly, the gene is expressed at
relatively low levels in young mice and mice that do not show symptoms of lupus nephritis.
Gene expression is elevated in mice with lupus nephritis, and is lower in mice that have been
successfully treated with rapamycin or anti-B7 antibodies. The observation that expression
levels return to normal when kidney function is normal indicates that elevated levels are related
to, and diagnostic of, disease progression. Blocking the function of these genes may inhibit or
retard disease progression. Expression levels can also to used to assess and compare
effectiveness of various therapeutic interventions.
Accordingly, the BFLP0169 nucleic acid sequences are useful for detecting the presence
of lupus nephritis in a subject. Elevated levels of BFLP0169 transcripts or polypeptides relative
to levels in control samples indicate the presence of lupus nephritis in the subject. BFLP0169
nucleic acid sequences can also be used to monitor the effectiveness of treatments for lupus
5

nephritis: a decrease in expression of BFLP0169 genes relative to levels in diseased treatments
demonstrates that the treatment is effective.
The BFLP0169 sequences can additionally be used to identify therapeutic agents for
treating or preventing lupus nephritis in a subject. For example, a BFLP0169 polypeptide can be
contacted with a test agent. Binding of the BFLP0169 polypeptide to the test agent reveals that
the test agent modulates BFLP0169 activity. The BFLP0169-binding agent can be further tested
to determine if it acts to promote or inhibit lupus symptoms in a test organism (e.g., a NZB X
NZW mouse). Inhibition of lupus symptoms reveals that the agent is useful for treating or
preventing lupus nephritis, or symptoms associated with lupus nephritis. Additional utilities are
disclosed herein.
A 5987 nucleotide sequence that includes a human BFLP0169 nucleic acid is shown in
Table 1 (SEQ ID NO: 1). The human sequence was identified as the human ortholog of a murine
gene whose expression is increased in a NZB X NZW mouse with lupus nephritis-like
symptoms.
Nucleotides 1-5259 of the sequence shown in Table 1 encode a polypeptide of 1753
amino acids, whose sequence is shown in Table 2 (SEQ ID NO:2).

BFLP0169-like nucleic acids and polypeptides of the invention (including those shown in
Table 1) are referred to herein as "BFLP0169 " nucleic acids and polypeptides.
A BFLP0169 nucleic acid, and the encoded polypeptide, according to the invention are
useful in a variety of applications and contexts.
BFLP0169 shows horaolgy to other proteins as shown in the BLAST results decribed in
Table 3. KIAA0169, MAGE: 3461492, and 3598686, and cDNA: FLJ21639 are all proteins
encoded from partial reading frames (expressed sequence tags (ESTs)) found in geaomic DNA.
Because BFLP0169 has homology to these proteins, it is also encoded from either ;an entire open
reading frame, or part of a larger open reading frame (EST).

Table 3: Blast Results for BFLP0169
Gene Index/Identifier Protein/Organism Length(aa) Identity(%) Positives(%) Expect
gi11136397ldbj|D79991.11 Homo sapiensmRNA forKIAA0169protein, partialcds 1745 1635/1739(94%) 1635/1739(94%) 0.0
gi|22046118|ref|XP 052725.6|(XM 052725) similar toKIAA0169 protein[Homo sapiens] 1767 1635/1743(93%) 1635/1743(93%) 0.0
gi|23618434|ref|XP 130085.21(XM 130085) similar toKIAA0169 protein[Homo sapiens] 1111 949/1111(85%) 962/1111(87%) 0.0

gi|13529308|gb|AA Unknown (protein 853 740/801 740/801 0.0
H05407.1IAAH05407 for (92%) (92%)
(BC005407) IMRGE:3461492)
[Homo sapiens]
gi|19343754|gb|AA Similar to 525 411/522 422/522 0.0
H25526.ll KIAA0169 protein (78%) (80%)
(BC025526) [Mus musculus]
Table 4 shows a ClustalW alignment of BFLP0169 (SEQ ID NO:2) against the proteins
described above in Table 3.
Table 4. ClustalW Analysis of SEQ ID NO:2
1) SEQ ID NO:2
2) gi11136397 Idb|ID79991.1l (SEQ ID NO:21)
3) gi|22046118|ref1XP_O52725.6| (XM_052725) (SEQ ID NO:22)
4) gi|23618434|ref|XP_130085.2| (XM_130085) (SEQ ID NO:23)
5) gi|13529308|gb|AAH05407.1|AAH05407 (BC005407) (SEQ ID NO:24)
6) gi|19343754|gb|AAH25526.1| (BC025526) (SEQ ID NO:25)

Residues 1-14 of SEQ ID NO:2 are referred to herein as SEQ ID NO:20. The fragment of
SEQ ID NO:21 that includes amino acids 1-6 is referred to herein as SEQ ID NO:26.
BFLP0169 Nucleic Acids
The nucleic acids of the invention include those that encode a BFLP0169 polypeptide or
protein. As used herein, the terms polypeptide and protein are interchangeable.
In some embodiments, a BFLP0169 nucleic acid encodes a mature BFLP0169
polypeptide. As used herein, a "mature" form of a polypeptide or protein described herein relates
to the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally
occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full
length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the
polypeptide, precursor or proprotein encoded by an open reading frame described herein. The
product "mature" form arises, again by way of nonlimiting example, as a result of one or more
naturally occurring processing steps that may take place within the cell in which the gene product
arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein
include the cleavage of the N-terrninal methionine residue encoded by the initiation codon of an
open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a
mature form arising from a precursor polypeptide or protein that has residues 1 to N, where
residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal
of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide
or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to
residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further
as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-
translational modification other than a proteolytic cleavage event. Such additional processes
include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In
13

general, a mature polypeptide or protein may result from the operation of only one of these
processes, or a combination of any of them.
The invention includes mutant or variant nucleic acids of SEQ ID NO: 1, or a fragment
thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID
NO:1, while still encoding a protein that maintains at least one of its BFLP0169 -like activities
and physiological functions (i.e., modulating angiogenesis, neuronal development). The
invention further includes the complement of the nucleic acid sequence of SEQ ID NO:1,
including fragments, derivatives, analogs and homologs thereof. The invention additionally
includes nucleic acids or nucleic acid fragments, or complements thereto, whose sti-uctures
include chemical modifications.
One aspect of the invention pertains to isolated nucleic acid molecules that encode
BFLP0169 proteins or biologically active portions thereof. Also included are nuckic acid
fragments sufficient for use as hybridization probes to identify BFLP0169 -encoding nucleic
acids (e.g., BFLP0169 mRNA) and fragments for use as polymerase chain reaction (PCR)
primers for the amplification or mutation of BFLP0169 nucleic acid molecules. As; used herein,
the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic
DNA), RNA molecules (eg:, mRNA), analogs of the DNA or RNA generated using nucleotide
analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is double-stranded DNA.
"Probes" refer to nucleic acid sequences of variable length, preferably between at least
about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes
are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer
length probes are usually obtained from a natural or recombinant source, are highly specific and
much slower to hybridize than oligomers. Probes may be single- or double-stranded and
designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like
technologies.
An "isolated" nucleic acid molecule is one that is separated from other nucleic acid
molecules that are present in the natural source of the nucleic acid. Examples of isolated nucleic
acid molecules include, but are not limited to, recombinant DNA molecules contained in a
vector, recombinant DNA molecules maintained in a heterologous host cell, partially or
14

substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably,
an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid {i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various embodiments, the isolated
BFLP0169 nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb,
1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in
genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated"
nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular
material or culture medium when produced by recombinant techniques, or of chemical precursors
or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO: 1, or a complement thereof, can be isolated using standard
molecular biology techniques and the sequence information provided herein. Using all or a
portion of the nucleic acid sequence of SEQ ID NO:1 as a hybridization probe, BFLP0169
nucleic acid sequences can be isolated using standard hybridization and cloning techniques {e.g.,
as described in Sambrook et ah, eds., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ah, eds.,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR
amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector
and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to
BFLP0169 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an
automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues,
which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA
sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or
complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions
of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to
15

30 nt in length. In one embodiment, an oligonucleotide comprising a nucleic acid molecule less
than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO:1,
or a complement thereof. Oligonucleotides may be chemically synthesized and may be used as
probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a
nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:1,
or a portion of this nucleotide sequence. A nucleic acid molecule that is complementary to the
nucleotide sequence shown in SEQ ID NO:1 is one that is sufficiently complementary to the
nucleotide sequence shown in SEQ ED NO:1 that it can hydrogen bond with little or no
mismatches to the nucleotide sequence shown in SEQ ID NO:1, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base
pairing between nucleotide units of a nucleic acid molecule, and the term "binding" means the
physical or chemical interaction between two polypeptides or compounds or associated
polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Van der
Waals, hydrophobio interactions, etc. A physical interaction can be either direct or indirect.
Indirect interactions may be through or due to the effects of another polypeptide or compound.
Direct binding refers to interactions that do not take place through, or due to, the effect of another
polypeptide or compound, but instead are without other substantial chemical intermediates.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the
nucleic acid sequence of SEQ ID NO:1, e.g., a fragment that can be used as a probe or primer, or
a fragment encoding a biologically active portion of BFLP0169 . Fragments provided herein are
defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino
acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for
specific recognition of an epitope in the case of amino acids, respectively, and are at most some
portion less than a full length sequence. Fragments may be derived from any contiguous portion
of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or
amino acid sequences formed from the native compounds either directly or by modification or
partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a
structure similar to, but not identical to, the native compound but differs from it in respect to
16

certain components or side chains. Analogs may be synthetic or from a different evolutionary
origin and may have a similar or opposite metabolic activity compared to wild type:.
Derivatives and analogs may be full length or other than full length, if the derivative or
analog contains a modified nucleic acid or amino acid, as described below. Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules
comprising regions that are substantially homologous to the nucleic acids or proteins of the
invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even
99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of
identical size or when compared to an aligned sequence in which the alignment is done by a
computer homology program known in the art, or whose encoding nucleic acid is capable of
hybridizing to the complement of a sequence encoding the aforementioned proteins under
stringent, moderately stringent, or low stringent conditions. An exemplary program is the Gap
program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer
Group, University Research Park, Madison, WI) using the default settings, which uses the
algorithm of Smith and Waterman.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode those sequences
coding for isoforms of a BFLP0169 polypeptide. Isoforms can be expressed in different tissues
of the same organism as a result of, for example, alternative splicing of RNA. Alternatively,
isoforms can be encoded by different genes. In the present invention, homologous nucleotide
sequences include nucleotide sequences encoding for a BFLP0169 polypeptide of species other
than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat,
rabbit, dog, cat, cow, horse, and other organisms. Homologous nucleotide sequences also
include, but are not limited to, naturally occurring allelic variations and mutations of the
nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however,
include the nucleotide sequence encoding human BFLP0169 protein. Homologous nucleic acid
sequences include those nucleic acid sequences that encode conservative amino acid
substitutions (see below) in SEQ ID NO:2, as well as a polypeptide having BFLP0169 activity.
17

Biological activities of the BFLP0169 proteins are described below. A homologous: amino acid
sequence does not encode the amino acid sequence of a human BFLP0169 polypeptide.
The nucleotide sequence determined from the cloning of the human BFLP0169 gene
allows for the generation of probes and primers designed for use in identifying and/or cloning
BFLP0169 homologues in other cell types, e.g., from other tissues, as well as BFLP0169
homologues from other mammals. The probe/primer typically comprises a substan tially purified
oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that
hybridizes under stringent conditions to at least about 12,25, 50, 100, 150,200,250, 300,350 or
400 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1; or an anti-sense
strand nucleotide sequence of SEQ ID NO: 1; or of a naturally occurring mutant of SEQ ID NO: 1.
Probes based on the human BFLP0169 nucleotide sequence can be used to detect
transcripts or genomic sequences encoding the same or homologous proteins. In various
embodiments, the probe further comprises a label group attached thereto, e.g., the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can
be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a
BFLP0169 protein, such as by measuring a level of a BFLP0169-encoding nucleic acid in a
sample of cells from a subject e.g., detecting BFLP0169 mRNA levels or determining whether a
genomic BFLP0169 gene has been mutated or deleted.
A "polypeptide having a biologically active portion of BFLP0169 " refers to polypeptides
exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the
present invention, including mature forms, as measured in a particular biological assay, with or
without dose dependency. A nucleic acid fragment encoding a "biologically active portion of
BFLP0169 " can be prepared by isolating a portion of SEQ ID NO:1 that encodes a polypeptide
having a BFLP0169 biological activity (biological activities of the BFLP0169 proteins are
described below), expressing the encoded portion of BFLP0169 protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded portion of BFLP0169.
The invention also provides polymorphic forms of BFLP0169 nucleic acid sequences as
well as methods of detecting polymorphic sequences in BFLP0169 sequences The: polymorphic
forms include genomic sequences corresponding to exons and/or introns associated with
BFLP0169.
18

Individuals carrying polymorphic alleles of the invention may be detected at either the
DNA, the RNA, or the protein level using a variety of techniques that are well known in the art.
The present methods usually employ pre-characterized polymorphisms. That is, the genotyping
location and nature of polymorphic forms present at a site have already been determined. The
availability of this information allows sets of probes to be designed for specific identification of
the known polymorphic forms.
The genomic DNA used for the diagnosis may be obtained from any nucleated cells of the
body, such as those present in peripheral blood, urine, saliva, buccal samples, surgical specimen,
and autopsy specimens. The DNA may be used directly or may be amplified enzymatically in
vitro through use of PCR or other in vitro amplification methods such as the ligase chain reaction
(LCR), strand displacement amplification (SDA), self-sustained sequence replication (3SR),
prior to mutation analysis.
The detection of polymorphisms in specific DNA sequences, can be accomplished by a
variety of methods including, but not limited to, restriction-fragment-length-polymorphism
detection based on allele-specific restriction-endonuclease cleavage, hybridization with allele-.
specific oligonucleotide probes, including immobilized oligonucleotides or oligonucleotide
arrays, allele-specific PCR, mismatch-repair detection (MRD), binding of MutS protein,
denaturing-gradient gel electrophoresis (DGGE), single-strand-conformation-polymorphism
detection, RNAase cleavage at mismatched base-pairs, chemical or enzymatic cleavage of
heteroduplex DNA, methods based on allele specific primer_extension, genetic bit analysis
(GBA), the oligonucleotide-ligation assay (OLA), the allele-specific ligation chain reaction
(LCR), gap-LCR, radioactive and/or fluorescent DNA sequencing using standard procedures well
known in the art, and peptide nucleic acid (PNA) assays.
BFLP0169 Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide
sequences shown in SEQ ID NO:1 due to the degeneracy of the genetic code. These nucleic
acids thus encode the same BFLP0169 protein as that encoded by the nucleotide ssquence shown
in SEQ ID NO:1, e.g., the polypeptide of SEQ ID NO:2. In another embodiment, an isolated
19

nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an
amino acid sequence shown in SEQ ID NO:2.
In addition to the human BFLPO169 nucleotide sequence shown in SEQ ID NO: 1. it will
be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes
in the amino acid sequences of BFLPO 169 may exist within a population {e.g., the human
population). Such genetic polymorphism in the BFLPO 169 gene may exist among individuals
within a population due to natural allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a
BFLPO 169 protein, preferably a mammalian BFLPO 169 protein. Such natural allelic variations
can typically result in 1-5% variance in the nucleotide sequence of the BFLPO 169 gene. Any and
all such nucleotide variations and resulting amino acid polymorphisms in BFLPO 169 that are the
result of natural allelic variation and that do not alter the functional activity of BFLP0169 are
intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding BFLPO 169 proteins from other species, and
thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NO:1 are
intended to be within the scope of the invention. Nucleic acid molecules corresponding to
natural allelic variants and homologues of the BFLP0169 cDNAs of the invention can be isolated
based on their homology to the human BFLPO 169 nucleic acids disclosed herein usiing the human
cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization
techniques under stringent hybridization conditions. For example, a soluble human BFLP0169
cDNA can be isolated based on its homology to human membrane-bound BFLPO 1(59. Likewise,
a membrane-bound human BFLPO 169 cDNA can be isolated based on its homology to soluble
human BFLPO 169.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is
at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO:1. In another embodiment, the
nucleic acid is at least 10,25, 50, 100,250,500 or 750 nucleotides in length. In another
embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region.
As used herein, the term "hybridizes under stringent conditions" is intended to describe
20

conditions for hybridization and washing under which nucleotide sequences at least 60%
homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding BFLP0169 proteins derived from species other
than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high
stringency hybridization with all or a portion of the particular human sequence as a probe using
methods well known in the art for nucleic acid hybridization and cloning.
Thus, the present invention also includes polynucleotides capable of hybridizing under reduced
stringency conditions, more preferably stringent conditions, and most preferably highly stringent
conditions, to polynucleotides described herein. Examples of stringency conditions are shown in
the table below, highly stringent conditions are those that are at least as stringent as, for example,
conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and
reduced stringency conditions are at least as stringent as, for example, conditions M-R.
Table 4. Stringency Conditions

Stringency Polynucleotide Hybrid Hybridization Wash Temperature and
Condition Hybrid Length (bp)1 Temperature and Buffer"
Buffer11
A DNA-.DNA ≥50 65°C;lxSSC-or- 65°C; 0.3xSSC
42°C; lxSSC, 50%
formaraide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
67°C; 0.3xSSC
C DNA:KNA ≥50 67°C;lxSSC-or-
45°C; lxSSC, 50%
formamide
D DNA:RNA <50 TD*; lxSSC TD*; lxSSC
70°C; 0.3xSSC
E KNA:RNA ≥50 70°C;lxSSC-or-
50°C; lxSSC, 50%
formamide
F KNA:RNA <50 V; lxSSC Tf*; lxSSC
65°C; lxSSC
21

Stringency Polynucleotide Hybrid Hybridization Wash Temperature and
Condition Hybrid Length (bp)1 Temperature andBuffer" Buffer"
G DNA:DNA ≥50 65°C;4xSSC-or-
42°C;4xSSC,50%
formamide
H DNA:DNA <50 TH*; 4xSSC TH*;4xSSC
67°C; lxSSC
I DNA:RNA ≥50 67°C; 4xSSC -or-
45°C; 4xSSC, 50%
formamide
J DNA:RNA <50 T,*; 4xSSC TJ*; 4xSSC
67°C;lxSSC
K RNA:RNA ≥50 70°C; 4xSSC -or-
50°C; 4xSSC, 50%
formamide
L RNA:RNA <50 TL*; 2xSSC TL*; 2xSSC
50°C; ;txSSC
M DNArDNA ≥50 50°C; 4xSSC -or-
40°C;6xSSC, 50%formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
55°C; 2xSSC
O DNA:RNA ≥50 55°C; 4xSSC -or-
42°C; 6xSSC, 50%
formamide
P DNArRNA <50 TP*; 6xSSC TP*; 6?;SSC
60cC; :»xSSC
Q RNA:RNA ≥50 60°C; 4xSSC -or-
45°C; 6xSSC, 50%
formamide
R RNA:RNA <50 TR*; 4xSSC TR*; 4:cSSC
1: The hybrid length is that anticipated for the hybridized region(s) of the hybridizing
polynucleotides. When hybridizing a poiynucleotide to a target poiynucleotide of unknown
22

sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When
polynucleotides of known sequence are hybridized, the hybrid length can be determined by
aligning the sequences of the polynucleotides and identifying the region or regions of optimal
sequence complementarity.
H: SSPE (lxSSPE is 0.15MNaCl, 10mMNaH2PO4, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC (lxSSC is 0.15MNaCl and 15mM sodium citrate) in the hybridization and
wash buffers; washes are performed for 15 minutes after hybridization is complete.
TB* - TR*: The hybridization temperature for hybrids anticipated to be less than 50 base
pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm
is determined according to the following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in
length, Tm(°C) = 81.5 + 16.6(log10Na4) + 0.41(%G+C) - (600/N), where N is the number of bases
in the hybrid, and Na+ is the concentration of sodium ions in the hybridization buffer (Na+ for
lxSSC = 0.165 M).
Preferably, each such hybridizing polynucleotide has a length that is at least 25% (more
preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of
the present invention to which it hybridizes, and has at least 60% sequence identity (more
preferably, at least 75% identity; most preferably at least 90% or 95% identity) wi th the
polynucleotide of the present invention to which it hybridizes, where sequence identity is
determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to
maximize overlap and identity while minimizing sequence gaps.
A non-limiting example of stringent hybridization conditions is hybridization in a high
salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%
Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is
followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid
molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID
NO:1 corresponds to a naturally occurring nucleic acid molecule. As used herein., a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
23

In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO:1, or fragments, analogs or
derivatives thereof, under conditions of moderate stringency is provided. A non-limiting
example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X
Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed
by one or more washes in IX SSC, 0.1% SDS at 37°C.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:1, or fragments, analogs or derivatives
thereof, under conditions of low stringency, is provided. A non-limiting example of low
stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured
salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in
2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
Conservativ mutaions
In addition to naturally-occurring allelic variants of the BFLP0169 sequence that may
exist in the population, the skilled artisan will further appreciate that changes can be introduced
by mutation into the nucleotide sequence of SEQ ID NO:1, thereby leading to changes in the
amino acid sequence of the encoded BFLP0169 protein, without altering the functional ability of
the BFLP0169 protein. For example, nucleotide substitutions leading to amino acid substitutions
at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:1. A
"non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of
BFLP0169 without altering the biological activity, whereas an "essential" amino acid residue is
required for biological activity. For example, altering amino acid residues that are conserved
among the BFLP0169 proteins of the present invention, is likely to result in loss of activity of the
BFLP0169 protein.
Another aspect of the invention pertains to nucleic acid molecules encoding BFLP0169
proteins that contain changes in amino acid residues that are not essential for activity. Such
BFLP0169 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological
activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence
24

encoding a protein, wherein the protein comprises an amino acid sequence at least about 75%
homologous to the amino acid sequence of SEQ ID NO:2. Preferably, the protein encoded by the
nucleic acid is at least about 80% homologous to SEQ ID NO:2, more preferably at least about
90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO:2.
An isolated nucleic acid molecule encoding a BFLP0169 protein homologous to the
protein of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of SEQ ID NO:1, such that one or more
amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the nucleotide sequence of SEQ ID NO:1 by standard
techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more predicted non-essential amino
acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain. Families of amino acid residues
having similar side chains have been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side chains {e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus,
a predicted nonessential amino acid residue in BFLP0169 is replaced with another amino acid
residue from the same side chain family. Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of a BFLP0169 coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for BFLP0169 biological activity to
identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1 the encoded
protein can be expressed by any recombinant technology known in the art and the activity of the
protein can be determined.
In one embodiment, a mutant BFLP0169 protein can be assayed for (1) the ability to form
proteurprotein interactions with other BFLPO169 proteins, other cell-surface proteins, or
biologically active portions thereof, (2) complex formation between a mutant BFLPO 169 protein
and a BFLPO 169 receptor; (3) the ability of a mutant BFLPO 169 protein to bind to an
25

intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the
ability to bind BFLP0169 protein; or (5) the ability to specifically bind an anti-BFLP0169 protein
antibody.
Antisense BFLP0169 Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that
are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:1, or fragments, analogs or derivatives thereof. An "antisense" nucleic
acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a
protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or
complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are
provided that comprise a sequence complementary to at least about 10,25, 50,100, 250 or 500
nucleotides or an entire BFLP0169 coding strand, or to only a portion thereof. Nucleic acid
molecules encoding fragments, homologs, derivatives and analogs of a BFLP0169 protein of
SEQ ID NO.2, or antisense nucleic acids complementary to a BFLP0169 nucleic acid sequence
of SEQ ID NO: 1 are additionally provided.
Tn one embodiment, an antisense nucleic acid molecule is antisense to a "coding region"
of the coding strand of a nucleotide sequence encoding BFLP0169. The term "coding region"
refers to the region of the nucleotide sequence comprising codons which are translated into
amino acid residues (e.g., the protein coding region of human BFLP0169 corresponds to SEQ ID
NO:2). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding BFLP0169. The term "noncoding
region" refers to 5' and 3' sequences which flank the coding region that are not translated into
amino acids {i.e., also referred to as 5'and 3' untranslated regions).
Given the coding strand sequences encoding BFLP0169 disclosed herein (e.g., SEQ ID
NO:1), antisense nucleic acids of the invention can be designed according to the rules of Watson
and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary
to the entire coding region of BFLP0169 mRNA, but more preferably is an oligonucleotide that is
antisense to only a portion of the coding or noncoding region of BFLP0169 mRNA. For
example, the antisense oligonucleotide can be complementary to the region surrounding the
26

translation start site of BFLP0169 mRNA. An antisense oligonucleotide can be, for example,
about 5, 10,15,20,25,30, 35,40,45 or 50'nucleotides in length. An antisense nucleic acid of
the invention can be constructed using chemical synthesis or enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid
include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethy]-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-isopentenyladcnine, 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-thioumcil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the
antisense nucleic acid can be produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the
inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a
subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or
genomic DNA encoding a BFLP0169 protein to thereby inhibit expression of the protein, e.g., by
inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid
molecule that binds to DNA duplexes, through specific interactions in the major groove of the
27

double helix. An example of a route of administration of antisense nucleic acid molecules of the
invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules
can be modified to target selected cells and then administered systemically. For example, for
systemic administration, antisense molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic
acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III
promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an
a-anomeric nucleic acid molecule. An oc-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary to the usual P-units, the
strands run parallel to each other. The antisense nucleic acid molecule can also comprise a
2'-o-methyiribonucleotide.
Such modifications include, by way of nonlimiting example, modified bases, and nucleic
acids whose sugar phosphate backbones are modified or derivatized. These modifications are
carried out at least in part to enhance the chemical stability of the modified nucleic acid, such
that they may be used, for example, as antisense binding nucleic acids in therapeutic applications
in a subject.
BFLP0169 Ribozymes and PNA moieties
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.
Thus, ribozymes (e.g., hammerhead ribozymes) can be used to catalytically cleave BFLP0169
mRNA transcripts to thereby inhibit translation of BFLP0169 mRNA. A ribozyme having
specificity for a BFLP0169-encoding nucleic acid can be designed based upon the nucleotide
sequence of a BFLP0169 DNA disclosed herein (i.e., SEQ ED NO:1). For example, a derivative
of a Tetrahymena L-19 FVS RNA can be constructed in which the nucleotide sequence of the
28

active site is complementary to the nucleotide sequence to be cleaved in a BFLP0169-encoding
mRNA. Alternatively, BFLP0169 mRNA can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules.
Alternatively, BFLP0169 gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the BFLP0169 (e.g., the BFLP0169
promoter and/or enhancers) to form triple helical structures that prevent transcription of the
BFLP0169 gene in target cells.
In various embodiments, the nucleic acids of BFLP0169 can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate backbone of trie nucleic
acids can be modified to generate peptide nucleic acids. As used herein, the terms "peptide
nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four
natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of
PNA oligomers can be performed using standard solid phase peptide synthesis protocols.
PNAs of BFLP0169 can be used in therapeutic and diagnostic applications. For example,
PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene
expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of
BFLP0169 can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g.,
PNA directed PCR clamping; as artificial restriction enzymes when used in combination with
other enzymes, e.g., SI nucleases; or as probes or primers for DNA sequence and hybridization.
In another embodiment, PNAs of BFLP0169 can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug
delivery known in the art. For example, PNA-DNA chimeras of BFLP0169 can be: generated that
may combine the advantageous properties of PNA and DNA.
29

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 or
the blood-brain barrier. In addition, oligonucleotides can be modified with hybridization
triggered cleavage agents or intercalating agents. To this end, the oligonucleotide may be
conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a
transport agent, a hybridization-triggered cleavage agent, etc.
BFLP0169 Interfering Nucleic Acids
Also provided by the invention is an isolated double-stranded nucleic acid (DNA or
RNA) that is capable of mediating specific inhibition of BFLP0169 gene expression. In
preferred embodiments, one or both strands of the double-stranded molecule is an RNA
molecule. Preferably, each RNA strand has a length from 19-25, particularly from 19-23
nucleotides, more particularly from 20-22 nucleotides, and is capable of mediating BFLP0169
target-specific nucleic acid modifications, particularly RNA interference and/or DNA
methylation. The double-stranded BFLP0169 molecule may be double stranded or have an
overhang at one or both the 5' and/or 3' terminus. For example, the molecule may have a 3'
overhang. The length of the 3'-overhang can be, e.g., 1-6 nucleotides, 2-5 nucleotides, 3-4
nucleotides, or 2 nucleotides. The length of the overhang may be the same or different for each
strand. In one embodiment, dsRNAs are composed of two 21 nucleotide strands that are paired
such that 1,2, or 3 nucleotide overhangs are present on both ends of the double-stranded RNA.
The RNA strands preferably have 3'-hydroxyl groups. The 5'-terminus preferably
includes a phosphate, diphosphate, triphosphate or hydroxyl group. If desired, the 3'-overhangs
may be stabilized against degradation. For example, they may be selected such that they consist
of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, pyrimidine
nucleotides may be replaced with modified analogues, e.g. substitution of undine -2 nucleotide
3' overhangs by 2'-deoxythymidine is tolerated, and does not affect the efficiency of RNA
interference. The RNA molecule may contain at least one modified nucleotide analogue. The
nucleotide analogues may be located at positions where the target-specific activity, e.g. the RNAi
mediating activity is not substantially affected. The modified nucleotide is preferably present in a
30

region at the 5'-end and/or the 3'-end of the double-stranded RNA molecule. In some
embodiments, overhangs are stabilized by incorporating modified nucleotide analogues.
Nucleotide analogues can include sugar- or backbone-modified ribonucleotides. Other
suitable nucleotides include a non-naturally occurring nucleobase instead of a naturally occurring
nucleobases. For example, analogues can include undines or cytidines modified at the 5-
position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified
at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; 0- and N-
alkylated nucleotides, e.g. N6-methyl adenosine are suitable. In preferred sugar-modified
ribonucleotides the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR,
NH2, NHR, NR2 or CN, wherein R is C1,-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. In
a preferred embodiment, where backbone-modified ribonucleotides are used as the phosphoester
group connecting to adjacent ribonucleotides, they are replaced by a modified group, e.g. a
phosphothioate group. It should be noted that the above modifications may be combined.
The BFLP0169 interfering RNA molecule can be a naturally isolated RNA molecule or
can a synthetic RNA molecule. Preferably, the BFLP0169 interfering RNA molecule is
substantially free from contaminants occurring in cell extracts, e.g. from Drosophila embryos.
Further, the BFLP0169 interfering RNA molecule is preferably substantially free from any non-
target-specific contaminants, particularly non-target-specific RNA molecules e.g. irom
contaminants occurring in cell extracts.
Isolated double-stranded BFLP0169 interfering molecules can be used for mediating
BFLP0169 target-specific nucleic acid modifications, particularly RNAi, in mammalian cells, .
particularly in human cells.
The sequence of the double-stranded BFLP0169 interfering molecule of the present
invention is of sufficient identity to a nucleic acid BFLP0169 target molecule in order to effect
target-specific interference of BFLP0169 gene expression and/or DNA methylation. Preferably,
the sequence has an identity of at least 50%, particularly of at least 70% to the desired target
molecule in the double-stranded portion of the RNA molecule. More preferably, the identity is at
least 85% and most preferably 100% in the double-stranded portion of the RNA molecule. The
identity of a BFLP0169 double-stranded interfering RNA molecule to a predetermined nucleic
31

acid target molecule, e.g. an BFLP0169 mRNA target molecule with the sequence shown in SEQ
ID NO:1, may be determined using the equation: I =(n/L) x 100, wherein I is the identity in
percent, n is the number of identical nucleotides in the double-stranded portion of the ds P.NA
and the target and L is the length of the sequence overlap of the double-stranded portion of the
dsRNA and the target.
Alternatively, the identity of the double-stranded RNA molecule relative to the target
sequence may also be defined including the 3' overhang, particularly an overhang having a length
from 1-3 nucleotides. In this case the sequence identity is preferably at least 50%, more
preferably at least 70% and most preferably at least 85% to the target sequence. For example, the
nucleotides from the 3' overhang and up to 2 nucleotides from the 5' and/or 3' terminus of the
double strand may be modified without significant loss of activity.
A double-stranded BFLP0169 RNA molecule may be prepared by a method that includes
synthesizing two RNA strands each having a length from 19-25, e.g. from 19-23 nucleotides,
wherein said RNA strands are capable of forming a double-stranded RNA molecule, wherein
preferably at least one strand has a 3'-overhang from 1-5 nucleotides, and (b) combining the
synthesized RNA strands under conditions, wherein a a double-stranded RNA molecule is
formed. The double-stranded RNA molecule is capable of mediating target-specific nucleic acid
modifications, particularly RNA interference and/or DNA methylation.
Methods of synthesizing RNA molecules are known in the art. The single-stranded RNAs
can also be prepared by enzymatic transcription from synthetic DNA templates or from DNA
plasmids isolated from recombinant bacteria. Typically, phage RNA polymerases Eire used such
as T7, T3 or SP6 RNA polymerase.
A further aspect of the present invention relates to a method of mediating EIFLP0169-
specific nucleic acid modifications, particularly RNA interference and/or DNA methylation in a
cell or an organism by contacting the cell or organism with the double-stranded RNA molecule
of the invention under conditions wherein target-specific nucleic acid modifications may occur
and mediating a target-specific nucleic acid modification effected by the double-stranded RNA
towards a BFLP0169 target nucleic acid.
32

BFLP0169 Polypeptides
A BFLP0169 polypeptide of the invention includes the BFLP0169-like protein whose
sequence is provided in SEQ ID NO:2. The invention also includes a mutant or variant form of
the disclosed BFLP0169 polypeptide, or of any of the fragments of the herein disclosed
BFLP0169 polypeptide sequences.
Thus, a BFLP0169 polypeptide includes one in which any residues may be changed from
the corresponding residue shown in SEQ ID NO:2 while still encoding a protein that maintains
its BFLP0169-like activities and physiological functions, or a functional fragment thereof. In
some embodiments, up to 20% or more of the residues may be so changed in the mutant or
variant protein. In some embodiments, the BFLP0169 polypeptide according to the: invention is a
mature polypeptide.
Rapamycin Binding Domains
To identify regions of aBFLP0169 polypeptide sequence (e.g., a polypeptide including all
or a portion of SEQ ID NO:2) containing rapamycin binding domains, the entire coding
sequence, or a fragment of a BFLP0169 polypeptide sequence, is tested for its ability to bind
rapamycin. Any technique known in the art for determining binding of a polypeptide to a small
molecule can be used. For example, rapamycin can be labeled {i.e., with a non-radioactive label
or with a radiolabel (e.g.,14C, 32P, 3H, or 125I), and mixed with a polypeptide containing some or
all of a BFLP0I69 polypeptide sequence. The polypeptide optionally includes a moiety that
facilitates detection, e.g., the polypeptide can be a fusion polypeptide that includes a BFLP0169
sequence and anon-BFLP0169 polypeptide sequence.
A reagent specific for the polypeptide containing the BFLP0169 polypeptide sequence
(e.g., an antibody specific for BFLP0169 or a probe specific for the non-BFLP0169 polypeptide
in the case of a fusion polypeptide) is added to the mixture. Complexes that bind to the reagent
are isolated, and the presence of label, which reveals the presence of rapamycin, is determined.
In general, a BFLP0169-like variant that preserves BFLP0169-like function includes any
variant in which residues at a particular position in the sequence have been substituted by other
33

amino acids, and further include the possibility of inserting an additional residue or residues
between two residues of the parent protein as well as the possibility of deleting one or more
residues from the parent sequence. Any amino acid substitution, inserticr. or deletion is
encompassed by the invention. In favorable circumstances, the substitution is a conservative
substitution as defined above.
One aspect of the invention pertains to isolated BFLP0169 proteins, and biologically
active portions thereof, or derivatives, fragments, analogs or homologs thereof. Fragments can
comprise contigous stretches of SEQ ID NO:2, or interspersed segments of SEQ ED NO:2. Also
provided are polypeptide fragments suitable for use as immunogens to raise anti-BFLP0169
antibodies. In one embodiment, native BFLP0169 proteins can be isolated from cells or tissue
sources by an appropriate purification scheme using standard protein purification techniques. In
another embodiment, BFLP0169 proteins are produced by recombinant DNA techniques.
Alternative to recombinant expression, a BFLP0169 protein or polypeptide can be synthesized
chemically using standard peptide synthesis techniques.
A "purified" protein or biologically active portion thereof is substantially free of cellular
material or other contaminating proteins from the cell or tissue source from which the BFLP0169
protein is derived, or substantially free from chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of cellular material" includes
preparations of BFLP0169 protein in which the protein is separated from cellular components of
the cells from which it is isolated or recombmantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of BFLP0169 protein having less
than about 30% (by dry weight) of non-BFLP0169 protein (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of non-BFLP0169 protein, still
more preferably less than about 10% of non-BFLP0169 protein, and most preferably less than
about 5% non-BFLP0169 protein. When the BFLP0169 protein or biologically active portion
thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less than about 10%, and most
preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes
preparations of BFLP0169 protein in which the protein is separated from chemical precursors or
34

other chemicals that are involved in the synthesis of the protein. In one embodiment, the
language "substantially free of chemical precursors or other chemicals" includes preparations of
BFLP0169 protein having less than about 30% (by dry weight) of chemical precursors or
non-BFLP0169 chemicals, more preferably less than about 20% chemical precursors or
non-BFLP0169 chemicals, still more preferably less than about 10% chemical precursors or
non-BFLP0169 chemicals, and most preferably less than about 5% chemical precursors or
non-BFLP0169 chemicals.
Biologically active portions of aBFLP0169 protein include peptides comprising amino
acid sequences sufficiently homologous to or derived from the amino acid sequence of the
BFLP0169 protein, e.g., the amino acid sequence shown in SEQ ID NO:2 that include fewer
amino acids than the full length BFLP0169 proteins, and exhibit at least one activity of a
BFLP0169 protein. Typically, biologically active portions comprise a domain or motif with at
least one activity of the BFLP0169 protein. A biologically active portion of a BFLP0169 protein
can be a polypeptide which is, for example, 10,25,50,100 or more amino acids in length.
A biologically active portion of a BFLP0169 protein of the present invention may contain
at least one of the above-identified domains conserved between the BFLP0169 proteins.
Moreover, other biologically active portions, in which other regions of the protein are deleted,
can be prepared by recombinant techniques and evaluated for one or more of the functional
activities of a native BFLP0169 protein.
In an embodiment, the BFLP0169 protein has an amino acid sequence sho\vn in SEQ ID
NO:2. In other embodiments, the BFLP0169 protein is substantially homologous to SEQ ID
NO:2 and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid
sequence due to natural allelic variation or mutagenesis, as described in detail below.
Accordingly, in another embodiment, the BFLP0169 protein is a protein that comprises an amino
acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2 and
retains the functional activity of the BFLP0169 proteins of SEQ IDNO:2.
Determining homology between two or more sequences
To determine the percent homology of two amino acid sequences or of two nucleic acid
sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be
35

introduced in either of the sequences being compared for optimal alignment between the
sequences). 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 nucleic acid sequence homology may be determined as the degree of identity between
two sequences. The homology may be determined using computer programs known in the art,
such as GAP software provided in the GCG program package. Using GCG GAP software with
the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and
GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred
to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%,
98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NO: 1.
The term "sequence identity" refers to the degree to which two polynucleotide or
poiypeptide sequences are identical on a residue-by-residue basis over a particular region of
comparison. The term "percentage of sequence identity" is calculated by comparing two
optimally aligned sequences over that region of comparison, determining the number of positions
at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids)
occurs in both sequences to yield the number of matched positions, dividing the number of
matched positions by the total number of positions rathe region of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of sequence identity. The term
"substantial identity" as used herein denotes a characteristic of a polynucleotide sequence,
wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity,
preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually
at least 99 percent sequence identity as compared to a reference sequence over a comparison
region. The term "percentage of positive residues" is calculated by comparing two optimally
aligned sequences over that region of comparison, determining the number of positions at which
the identical and conservative amino acid substitutions, as defined above, occur in both
sequences to yield the number of matched positions, dividing the number of matched positions
36

by the total number of positions in the region of comparison (i.e., the window size),, and
multiplying the result by 100 to yield the percentage of positive residues.
Chimeric and fusion proteins
The invention also provides BFLP0169 chimeric or fusion proteins. As used herein, a
BFLP0169 "chimeric protein" or "fusion protein" comprises a BFLP0169 polypeptide operatively
linked to a non-BFLP0169 polypeptide. A "BFLP0169 polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to BFLP0169, whereas a "non-BFLP0169
polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein
that is not substantially homologous to the BFLP0169 protein, e.g., a protein that is different
from the BFLP0169 protein and that is derived from the same or a different organism. Within a
BFLP0169 fusion protein the BFLP0169 polypeptide can correspond to all or a portion of a
BFLP0169 protein. An example of a BFLP0169 fusion polypeptide is one that includes amino
acids 21-230 of SEQ ID NO:2 (e.g., a polypeptide that includes amino acids 1-246 or amino
acids 21-246 of SEQ ID NO.2). In one embodiment, a BFLP0169 fusion protein comprises at
least one biologically active portion of a BFLP0169 protein. In another embodiment, a
BFLP0169 fusion protein comprises at least two biologically active portions of a EiFLP0169
protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the
BFLP0169 polypeptide and the non-BFLP0169 polypeptide are fused in-frame to each other.
The non-BFLP0169 polypeptide can be fused to the N-terminus or C-terminus of the BFLP0169
polypeptide.
For example, in one embodiment a BFLP0169 fusion protein comprises a BFLP0169
polypeptide operably linked to either an extracellular domain of a second protein, i.e., non-
BFLP0169 protein, or to the transmembrane and intracellular domain of a second protein, i.e.,
non-BFLP0169 protein. Such fusion proteins can be further utilized in screening assays for
compounds that modulate BFLP0169 activity (such assays are described in detail below).
In another embodiment, the fusion protein is a GST-BFLP0169 fusion protein in which
the BFLP0169 sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant BFLP0169.
37

In another embodiment, the fusion protein is a BFLP0169-immunoglobulin fusion protein
in which the BFLP0169 sequences comprising one or more domains are fused to sequences
derived from a member of the immunoglobulin protein family.
Inhibition of the BFLP0169 ligand/BFLP0169 interaction can be used therapeutically for
both the treatment of proliferative and differentiative disorders, e.g., cancer, modulating (e.g.,
promoting or inhibiting) cell survival as well as immunomodulatory disorders, autoimmunity,
transplantation, and inflammation by alteration of cyotokine and chemokine cascade
mechanisms. Moreover, the BFLP0169-immunoglobulin fusion proteins of the invention can be
used as immunogens to produce anti-BFLP0169 antibodies in a subject, to purify EFLP0169
ligands, and in screening assays to identify molecules that inhibit the interaction of BFLP0169
with aBFLP0169 ligand.
A BFLP0169 chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance with conventional techniques,
e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline
phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another
embodiment, the fusion gene can be synthesized by conventional techniques including automated
DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using
anchor primers that give rise to complementary overhangs between two consecutive gene
fragments that can subsequently be annealed and reamplifled to generate a chimeric gene
sequence. Moreover, many expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST polypeptide). A BFLP0169-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked in-frame to the BFLP0169
protein.
If desired, libraries of fragments of the BFLP0169 protein coding sequence: can be used to
generate a variegated population of BFLP0169 fragments for screening and subsequent selection
of variants of a BFLP0169 protein.
BFLP0169 Antibodies
38

Also included in the invention are antibodies to BFLP0169 proteins, or frapients of
BFLP0169 proteins. The term "antibody" as used herein refers to immunoglobulin molecules
and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that
contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab-
and F(ab')2 fragments, and an Fab expression library. In general, an antibody molecule obtained
from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one
another by the nature of the heavy chain present in the molecule. Certain classes have subclasses
as well, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa
chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes,
subclasses and types of human antibody species.
An isolated BFLP0169-related protein of the invention may be intended to serve as an
antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to
generate antibodies that immunospecifically bind the antigen, using standard techniques for
polyclonal and monoclonal antibody preparation. The full-length protein can be used or,
alternatively, the invention provides antigenic peptide fragments of the antigen for use as
immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the
amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID
NO:2, and encompasses an epitope thereof such that an antibody raised against the peptide forms
a specific immune complex with the full length protein or with any fragment that contains the
epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least
15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are
located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the
antigenic peptide is a region of BFLP0169-related protein that is located on the surface of the
protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human BFLPO 169-related
protein sequence will indicate which regions of a BFLPO 169-related protein are particularly
hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody
39

production. As a means for targeting antibody production, hydropathy plots showing regions of
hydrophilicity and hydrophobicity may be generated by any method well known in the art,
including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without
Fourier transformation. A Kylte & Doolitle plot was generated for the BFLPO169 protein, and is
shown in Table 5 below.
Table 5. Kyte & Doolittle Plot for BFLP0169

The novel nucleic acid encoding the BFLPO 169 protein of the invention, or fragments
thereof, may further be useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the protein are to be assessed. These materials are further useful in the generation
of antibodies that bind immunospecifically to the novel substances of the invention for use in
therapeutic or diagnostic methods. The disclosed BFLPO 169 protein has multiple hydrophilic
regions, each of which can be used as an immunogen. In one embodiment, a contemplated
BFLP0169 epitope is from about amino acids 20 to 90. In another embodiment, a BFLP0169
epitope is from about amino acids 100 to 130. In additional embodiments, BFLPO 169 epitopes
are from about amino acids 140 to 220, from about amino acids 240 to 250, from about amino
acids 280 to 290, from about amino acids 330 to 340, from about amino acids 370 to 380, from
about amino acids 400 to 410, from about amino acids 450 to 520, from about amino acids 530
to 540, from about amino acids 640 to 650, from about amino acids 720 to 730, from about
amino acids 800 to 820, from about amino acids 850 to 855, from about amino acids 900 to 910,
40

from about amino acids 920 to 930, from about amino acids 940 to 950, from about amino acids
970 to 990, from about amino acids 1000 to 1030, from about amino acids 1060 to 1080, from
about amino acids 1100 to 1110, from about amino acids 1170 to 1180, from about amino acids
1190 to 1210, from about amino acids 1250 to 1280, from about amino acids 1310 to 1320, from
about amino acids 1350 to 1370, from about amino acids 1400 to 1420, from about amino acids
1430 to 1440, from about amino acids 1500 to 1560, from about amino acids 1600 to 1610, from
about amino acids 1650 to 1690, from about amino acids 1700 to 1710, and from about amino
acids 1720 to 1730.
Antibodies that are specific for one or more domains within an antigenic protein, or
derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof,
may be utilized as an immunogen in the generation of antibodies that immunospecifically bind
these protein components.
Various procedures known within the art may be used for the production of polyclonal or
monoclonal antibodies directed against a protein of the invention, or against derivatives,
fragments, analogs homologs or orthologs thereof. The term "monoclonal antibody" (MAb) or
"monoclonal antibody composition", as used herein, refers to a population of antibody molecules
that contain only one molecular species of antibody molecule consisting of a unique light chain
gene product and a unique heavy chain gene product.
The antibodies directed against the protein antigens of the invention can further comprise
humanized antibodies or human antibodies. The humanized forms of antibodies include
chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the
sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human
immunoglobulin.
The antibodies can also be human antibodies, e.g., antibody molecules in which
essentially the entire sequences of both the light chain and the heavy chain, including the CDRs,
41

arise from human genes. Human monoclonal antibodies can be prepared by the tricma
technique; the human B-cell hybridoma technique and the EBV hybridoma technique.
Human antibodies can also be produced using phage display libraries, or by introducing
human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated. .Human antibodies may
additionally be produced using transgenic nonhuman animals that are modified so as to produce
fully human antibodies rather than the animal's endogenous antibodies in response to challenge
by an antigen.
The invention also provides single-chain antibodies specific to an antigenic protein of the
invention, In addition, methods can be adapted for the construction of Fab expression libraries to
allow rapid and effective identification of monoclonal Fab fragments with the desired specificity
for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that
contain the idiotypes to a protein antigen may be produced by techniques known in the art
including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody
molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab)2 fragment;
(iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a
reducing agent and (iv) Fv fragments.
Also provided by the invention are bispecific antibodies. Bispecific antibodies are
monoclonal, preferably human or humanized, antibodies that have binding specificities for at
least two different antigens. One of the binding specificities is for an antigenic protein of the
invention. The second binding target is any other antigen, and advantageously is a cell-surface
protein or receptor or receptor subunit.
If desired, antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least
part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant
region (CHI) containing the site necessary for light-chain binding present in at least one of the
fusions.
42

Bispecific antibodies can be provided as full length antibodies or antibody fragments
(e.g. F(ab')2 bispecific antibodies).
Also within the invention are antibodies with more than two valencies (such as trispecific
antibodies).
Exemplary bispecific antibodies bind to two different epitopes, at least one of which
originates in the protein antigen of the invention.
The invention also includes heteroconjugate antibodies, which include two covalently
joined antibodies.
The antibody of the invention can be modified to alter (e.g., enhance or diminish) its
function. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing
interchain disulfide bond formation in this region. The invention also includes
immunoconjugates that include an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or
animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Enzymatically active toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii
proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are
available for the production of radioconjugated antibodies. Examples include 212Bi, 1311,13IIn,
"Y, and 186Re.
The antibody can be conjugated to a "receptor" (such streptavidin) for utilization in rumor
pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by
removal of unbound conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
43

BFLP0169 Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors,
containing a nucleic acid encoding a BFLP0169 protein, or derivatives, fragments, analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated
into the viral genome. Certain vectors are capable of autonomous replication in a host cell into
which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into the host cell, and thereby are
replicated along with the host genomic sequence into which they have integreated. Moreover,
certain vectors are capable of directing the expression of genes to which they are operatively-
linked. Such vectors are referred to herein as "expression vectors". "Plasmid" and "vector" can
be used interchangeably as the plasmid is the most commonly used form of vector. However, the
invention is intended to include such other forms of expression vectors, such as viral vectors
(e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve
equivalent functions.
Within a recombinant expression vector, "operably-linked" is intended to mean that the
nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for
expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a
host cell when the vector is introduced into the host cell).The term "regulatory sequence" is
intended to include promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those that direct constitutive expression
of a nucleotide sequence in many types of host cell and those that direct expression of the
nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). The
expression vectors of the invention can be introduced into host cells to thereby produce proteins
or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein
(e.g., BFLP0169 proteins, mutant forms of BFLP0169 proteins, fusion proteins, etc.).
44

The recombinant expression vectors of the invention can be designed for expression of
BFLP0169 proteins in prokaryotic or eukaryotic cells. For example, BFLP0169 proteins can be
expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression
vectors) yeast cells or mammalian cells. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian
cells using a mammalian expression vector. Examples of mammalian expression vectors include
pCDM8 and pMT2PC. When used in mammalian cells, the expression vector's control functions
are often provided by viral regulatory elements. For example, commonly used promoters are
derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable
expression systems for both prokaryotic and eukaryotic cells.
In another embodiment, the recombinant mammalian expression vector is capable of
directing expression of the nucleic acid preferentially in a particular cell type {e.g., tissue-specific
regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are
known in the art. Non-limiting examples of suitable tissue-specific promoters include the
albumin promoter (liver-specific), lymphoid-specific promoters, in particular promoters of T cell
receptors and immunoglobulins, neuron-specific promoters {e.g., the neurofilament promoter),
pancreas-specific promoters, and mammary gland-specific promoters (e.g., milk whey promoter).
Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters and
the oc-fetoprotein promoter.
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation. That is,
the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for
expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to
BFLP0169 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen that direct the continuous expression of the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of
45

antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a
high efficiency regulatory region, the activity of which can be determined by the cell type into
which the vector is introduced.
Another aspect of the invention pertains to host cells into which a recombinant expression
vector of the invention has been introduced. The terms "host cell" and "recombinant host cell"
are used interchangeably herein. It is understood that such terms refer not only to the particular
subject cell but also to the progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either mutation or environmental
influences, such progeny may not, in fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, BFLP0169 protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as
human, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to
those skilled in the art.
A gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally
introduced into the host cells along with the gene of interest. Various selectable markers include
those that confer resistance to drugs, such as G418, hygromycin and methotrexate. A nucleic
acid encoding a selectable marker can be introduced into a host cell on the same vector as that
encoding BFLP0169 or can be introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the
selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can
be used to produce (i.e., express) BFLP0169 protein. Accordingly, the invention further provides
methods for producing BFLP0169 protein using the host cells of the invention. In one
embodiment, the method comprises culturing the host cell of invention (into which a
recombinant expression vector encoding BFLP0169 protein has been introduced) in a suitable
46

medium such that BFLP0169 protein is produced. In another embodiment, the method further
comprises isolating BFLP0169 protein from the medium or the host cell.
Transgenic BFLP0169 Animals
The host cells of the invention can also be used to produce non-human transgenic
animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an
embryonic stem cell into which BFLP0169 protein-coding sequences have been introduced. Such
host cells can then be used to create non-human transgenic animals in which exogenous
BFLP0169 sequences have been introduced into their genome or homologous recombinant
animals in which endogenous BFLP0169 sequences have been altered. Such animals are useful
for studying the function and/or activity of BFLP0169 protein and for identifying and/or
evaluating modulators of BFLP0169 protein activity. As used herein, a "transgenic animal" is a
non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in
which one or more of the cells of the animal includes a transgene. Other examples of transgenic
animais include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A
transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic
animal develops and that remains in the genome of the mature animal, thereby directing the
expression of an encoded gene product in one or more cell types or tissues of the transgenic
animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably
a mammal, more preferably a mouse, in which an endogenous BFLP0169 gene has been altered
by homologous recombination between the endogenous gene and an exogenous DNA molecule
introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development
of the animal.
A transgenic animal of the invention can be created by introducing BFLP0169-encoding
nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral
infection) and allowing the oocyte to develop in a pseudopregnant female foster animal.
Sequences including SEQ ID NO:1 can be introduced as a transgene into the genome of a
non-human animal. Alternatively, a non-human homologue of the human BFLP0169 gene, such
as a mouse BFLP0169 gene, can be isolated based on hybridization to the human BFLP0169
cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation
47

signals can also be included in the transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the BPLP0169
transgene to direct expression of BFLP0169 protein to particular cells. Methods for generating
transgenic animals via embryo manipulation and microinjection, particularly animals such as
mice, have become conventional in the art. Similar methods are used for production of other
transgenic animals. A transgenic founder animal can be identified based upon the presence of the
BFLP0169 transgene in its genome and/or expression of BFLP0169 mRNA in tissues or cells of
the animals. A transgenic founder animal can then be used to breed additional animals carrying
the transgene. Moreover, transgenic animals carrying a transgene-encoding BFLP0169 protein
can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least
a portion of a BFLP0169 gene into which a deletion, addition or substitution has been introduced
to thereby alter, e.g., functionally disrupt, the BFLP0169 gene. The BFLP0169 gene can be a
human gene.(e.g., the DNA of SEQ ID NO:1), but more preferably, is anon-humari homologue
of ahumanBFLP0169 gene. For example, a mouse homologue of human BFLP0169 gene of
SEQ ID NO: 1 can be used to construct a homologous recombination vector suitable for altering
an endogenous BFLP0169 gene in the mouse genome. In one embodiment, the vector is
designed such that, upon homologous recombination, the endogenous BFLP0169 gene is
functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock
out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the
endogenous BFLP0169 gene is mutated or otherwise altered but still encodes functional protein
(e.g., the upstream regulatory region can be altered to thereby alter the expression of the
endogenous BFLP0169 protein). In the homologous recombination vector, the altered portion of
the BFLP0169 gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the
BFLP0169 gene to allow for homologous recombination to occur between the exogenous
BFLP0169 gene carried by the vector and an endogenous BFLP0169 gene in an embryonic stem
cell. The additional flanking BFLP0169 nucleic acid is of sufficient length for successful
homologous recombination with the endogenous gene. Typically, several kilobases of flanking
48

DNA (both at the 5'- and 3'-termini) are included in the vector. The vector is then introduced
into an embryonic stem cell line (e.g., by electroporation) and cells in which the innoduced
BFLP0169 gene has homologously-recombined with the endogenous BFLP0169 gene are
selected.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form
aggregation chimeras. A chimeric embryo can then be implanted into a suitable pseudopregnant
female foster animal and the embryo brought to term. Progeny harboring the homologously-
recombined DNA in their germ cells can be used to breed animals in which all cells of the animal
contain the homologously-recombined DNA by germline transmission of the transgene.
In another embodiment, transgenic non-humans animals can be produced that contain
selected systems that allow for regulated expression of the transgene. One example of such a
system is the cre/loxP recombinase system of bacteriophage PI. For a description of the cre/loxP
recombinase system. Another example of a recombinase system is the FLP recombinase system
of Saccharnmyces cerevisiae. If a cre/loxP recombinase system is used to regulate; expression of
the transgene, animals containing transgenes encoding both the Cre recombinase and a selected
protein are required. Such animals can be provided through the construction of "double"
transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced
according to the methods described in the art. In brief, a cell (e.g., a somatic cell) from the
transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The
quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte
from an animal of the same species from which the quiescent cell is isolated. The reconstructed
oocyte is then cultured such that it develops to morula or blastocyte and then transferred to
pseudopregnant female foster animal. The offspring borne of this female foster animal will be a
clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Methods of Detecting BFLP0169 Nucleic Acids and Diagnosing Lupus Nephritis
49

Reagents that detect BFLP0169 nucleic acids and/or polypeptides can be used to detect
levels of BFLP0169 RNA and/or proteins sequences in a sample. Because elevated levels of
BFLP0169 RNA are found in anima).with lupus nephritis, detection of enhanced levels of
BFLP0169 RNA and/or BFLP0169 polypeptides indicates the presence or predisposition to lupus
in the subject. In addition, lowered levels of BFLP0169 RNA in treated lupus subjects as
compared to untreated lupus indicates a return to a non-lupus state. Thus, the efficacy of lupus
treatment can be monitored by comparing BFLP0169 RNA or protein levels in a sample from a
treated population to samples in a diseased but untreated sample, (or a sample from an individual
that has been treated for a shorter period of time).
Levels of BFLP0169 RNA can be assessed by comparing levels in a test cell population,
from a subject whose lupus status is unknown, to levels in a reference cell population whose
lupus status is known. Thus, the test cell population will typically include at least one cell that is
capable of expressing a BFLP0169 gene. By "capable of expressing" is meant that the gene is
present in an intact form in the cell and can be expressed. Expression of the BFLP0169 sequence
is then detected, if present, and, preferably, measured using methods known in the art. For
example, the BFLP0169 sequences disclosed herein can be used to construct probes for detecting
BFLP0169 RNA sequences in, e.g., northern blot hybridization analyses or methods which
specifically, and, preferably, quantitatively amplify BFLP0169 specific nucleic acid sequences.
Alternatively, the sequences can be used to construct primers for specifically ampl ifying the
BFLP0169 sequences in, e.g., amplification-based detection methods such as reverse-
transcription based polymerase chain reaction.
BFLP0169 expression can be also measured at the protein level, i.e., by measuring the
levels of BFLP0169 polypeptides. Such methods are well known in the art and include, e.g.,
immunoassays based on antibodies to proteins encoded by the genes.
Expression of sequences in test and control populations of cells can be compared using
any art-recognized method for comparing expression of nucleic acid sequences. V/hether or not
comparison of the gene expression profile in the test cell population to the reference cell
population reveals the presence, or degree, of the measured parameter depends on the
composition of the reference cell population. For example, if the reference cell population is
composed of cells from a lupus free subject, a similar gene expression level in the test cell
50

population and a reference cell population indicates the test cell population is from a lupus free
subject.. Conversely, if the reference cell population is made up of cells from a diseased subject,
a similar gene expression profile between the test cell population and the reference cell
population indicates the test cell population is from a subject with lupus.
In various embodiments, a BFLP0169 sequence in a test cell population is considered
comparable in expression level to the expression level of the ADIPO sequence in the reference
cell population if its expression level varies within a factor of 2.0,1.5, or 1.0 fold to the level of
the BFLP0169 transcript in the reference cell population. In various embodiments., a BFLP0169
sequence in a test cell population can be considered altered in levels of expression if its
expression level varies from the reference cell population by more than 1.0, 1.5,2.0 or more fold
from the expression level of the corresponding BFLP0169 sequence in the reference cell
population.
If desired, comparison of differentially expressed sequences between a test cell
population and a reference cell population can be done with respect to a control nucleic acid
whose expression is independent of the parameter or condition being measured. Expression
levels of the control nucleic acid in the test and reference nucleic acid can be used to normalize
signal levels in the compared populations. Suitable control nucleic acids can readily be
determined by one of ordinary skill in the art.
In some embodiments, the test cell population is compared to multiple reference cell
populations. Each of the multiple reference populations may differ in the known parameter.
Thus, a test cell population may be compared to a first reference cell population from a subject
known to have lupus, as well as a second reference population known to not have lupus.
The test cell population that is exposed can be any number of cells, i.e., one or more cells,
and can be provided in vitro, in vivo, or ex vivo.
Preferably, cells in the reference cell population are derived from a tissue type as similar
as possible to test cell, e.g., renal tissue. In some embodiments, the control cell is derived from
the same subject as the test cell. In other embodiments, the reference cell population is derived
51

from a plurality of cells from multiple subjects. For example, the reference cell population can
be a database of expression patterns from previously tested cells.
The subject is preferably a mammal. The mammal can be, e.g., a human, non-human
primate, mouse, rat, dog, cat, horse, or cow.
Pharmaceutical Compositions
The BFLP0169 nucleic acid molecules, BFLP0169 proteins, and anti-BFLP0169
antibodies (also referred to herein as "active compounds") of the invention, and derivatives,
fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions
suitable for administration. Such compositions typically comprise the nucleic acid molecule,
protein, or antibody and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and
the like, compatible with pharmaceutical administration. Suitable carriers are described in the
most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the
field, which is incorporated herein by reference. Preferred examples of such carriers or diluents
include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5%
human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
The use of such media and agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated. Supplementary active; compounds
can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its
intended route of administration. Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),
transmucosal, and rectal administration.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions
(where water soluble) or dispersions and sterile powders for the extemporaneous preparation of
sterile injectable solutions or dispersion.
52

Dosage unit form as used herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated
by and directly dependent on the unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an
active compound for the treatment of individuals.
Sustained-release preparations can be prepared. Suitable examples of sustained-release
preparations include semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-
hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid
and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic
acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybuiyric acid.
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of
molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
The pharmaceutical compositions can be included in a container, pack, or dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express BFLP0169
protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to
detect BFLP0169 mRNA (e.g., in a biological sample) or a genetic lesion in a BFLP0169 gene,
and to modulate BFLP0169 activity, as described further, below. In addition, the BFLP0169
proteins can be used to screen drugs or compounds that modulate the BFLP0169 protein activity
or expression as well as to treat disorders characterized by insufficient or excessive production of
BFLP0169 protein or production of BFLP0169 protein forms that have decreased or aberrant
activity compared to BFLP0169 wild-type protein. In addition, the anti-BFLP0169 antibodies of
53

the invention can be used to detect and isolate BFLP0169 proteins and modulate BFLP0169
activity. For example, BFLP0169 activity includes T-cell or NK cell growth and differentiation,
antibody production, and tumor growth.
The invention further pertains to novel agents identified by the screening assays described
herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for
identifying modulators, i.e., candidate or test compounds or agents {e.g., peptides,
peptidomimetics, small molecules or other drugs) that bind to BFLP0169 proteins or have a
stimulatory or inhibitory effect on, e.g., BFLP0169 protein expression or BFLP0169 protein
activity. The invention also includes compounds identified in the screening assays described
herein.
In one embodiment, the screening assays are used to identify therapeutic agents for
treating autoimmune diseases. The autoimmune disease can be, e.g., lupus, including lupus
nephtitis.
In one embodiment, the invention provides assays for screening candidate or test
compounds which bind to or modulate the activity of the membrane-bound form of a BFLP0169
protein or polypeptide or biologically-active portion thereof. The test compounds of the
invention can be obtained using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; spatially addressable parallel solid
phase or solution phase libraries; synthetic library methods requiring deconvolution; the
"one-bead one-compound" library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is limited to peptide libraries, while
the other four approaches are applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds.
A "small molecule" as used herein, is meant to refer to a composition that lias a molecular
weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can
be, e.g., rapamycin,nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids
54

or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such
as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the
assays of the invention. The libraries of compounds may be presented in solution, or on beads,
on chips, bacteria, spores, plasmids or on phage
In one embodiment, an assay is a cell-based assay in which a cell which expresses a
membrane-bound form of BFLP0169 protein, or a biologically-active portion thereof, on the cell
surface is contacted with a test compound and the ability of the test compound to bind to a
BFLP0169 protein determined. The cell, for example, can be of mammalian origin, or a yeast
cell. Determining the ability of the test compound to bind to the BFLP0169 protein can be
accomplished, for example, by coupling the test compound with a radioisotope or enzymatic
label such that binding of the test compound to the BFLP0169 protein or biologically-active
portion thereof can be determined by detecting the labeled compound in a complex. For
example, test compounds can be labeled with 1251,3SS, 14C, or 3H, either directly or indirectly,
and the radioisotope detected by direct counting of radioemission or by scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish
peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to product. In one embodiment, the
assay comprises contacting a cell which expresses a membrane-bound form of BFLP0169
protein, or a biologically-active portion thereof, on the cell surface with a known compound
which binds BFLP0169 to form an assay mixture, contacting the assay mixture wilh a test
compound, and determining the ability of the test compound to interact with a BFLP0169
protein, wherein determining the ability of the test compound to interact with a BFLP0169
protein comprises determining the ability of the test compound to preferentially bind to
BFLP0169 protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell
expressing a membrane-bound form of BFLP0169 protein, or a biologically-active: portion
thereof, on the cell surface with a test compound and determining the ability of the: test
compound to modulate (e.g., stimulate or inhibit) the activity of the BFLP0169 protein or
biologically-active portion thereof. Determining the ability of the test compound to modulate the
55

activity of BFLP0169 or a biologically-active portion thereof can be accomplished, for example,
by determining the ability of the BFLP0169 protein to bind to or interact with a BFLP0169 target
molecule. As used herein, a "target molecule" is a molecule with which a BFLP0169 protein
binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a
BFLP0169 interacting protein, a molecule on the surface of a second cell, a molecule in the
extracellular milieu, a molecule associated with the internal surface of a cell membrane or a
cytoplasmic molecule. A BFLP0169 target molecule can be a non-BFLP0169 molecule or a
BFLP0169 protein or polypeptide of the invention In one embodiment, a BFLP0169 target
molecule is a component of a signal transduction pathway that facilitates transduction of an
extracellular signal {e.g. a signal generated by binding of a compound to a membrane-bound
BFLP0169 molecule) through the cell membrane and into the cell. The target, for example, can
be a second intercellular protein that has catalytic activity or a protein that facilitates the
association of downstream signaling molecules with BFLP0169.
Determining the ability of the BFLP0169 protein to bind to or interact with a BFLP0169
target molecule can be accomplished by one of the methods described above for determining
direct binding. In one embodiment, determining the ability of the BFLP0169 protein to bind to or
interact with a BFLP0169 target molecule can be accomplished by determining the activity of the
target molecule. For example, the activity of the target molecule can be determined by detecting
induction of a cellular second messenger of the target (i.e. intracellular Ca2+, diacylglycerol, IP3,
etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the
induction of a reporter gene (comprising a BFLP0169-responsive regulatory element operatively
linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting; a cellular
response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising
contacting a BFLP0169 protein or biologically-active portion thereof with a test compound and
determining the ability of the test compound to bind to the BFLP0169 protein or biologically-
active portion thereof. Binding of the test compound to the BFLP0169 protein can be determined
either directly or indirectly as described above. In one such embodiment, the assay comprises
contacting the BFLP0169 protein or biologically-active portion thereof with a known compound
56

which binds BFLP0169 to form an assay mixture, contacting the assay mixture with a test
compound, and determining the ability of the test compound to interact with a BFLP0169
protein, wherein determining the ability of the test compound to interact with a BFLPO169
protein comprises determining the ability of the test compound to preferentially bind to
BFLPO 169 or a biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting
BFLP0169 protein or a biologically-active portion thereof with a test compound and determining
the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the
BFLPO 169 protein or a biologically-active portion thereof. Determining the ability of the test
compound to modulate the activity of BFLPO 169 can be accomplished, for example, by
determining the ability of the BFLPO 169 protein to bind to a BFLP0169 target molecule by one
of the methods described above for determining direct binding. In an alternative embodiment,
determining the ability of the test compound to modulate the activity of BFLPO 169 protein can
be accomplished by determining the ability of the BFLPO 169 protein further modulate a
BFLP0169 target molecule. For example, the catalytic/enzymatic activity of the target molecule
on an appropriate substrate can be determined as described above.
In yet another embodiment, the cell-free assay comprises contacting the BFLP0169
protein or a biologically-active portion thereof with a known compound which binds BFLPO 169
protein to form an assay mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to interact with a BFLP0169 protein, wherein
determining the ability of the test compound to interact with a BFLP0169 protein comprises
determining the ability of the BFLP0169 protein to preferentially bind to or modulate the activity
of a BFLPO 169 target molecule.
The cell-free assays of the invention are amenable for use with both the soluble form or
the membrane-bound form of BFLPO 169 protein. In the case of cell-free assays comprising the
membrane-bound form of BFLPO 169 protein, it may be desirable to utilize a solubilizing agent
such that the membrane-bound form of BFLP0169 protein is maintained in solution. Examples
of such solubilizing agents include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
57

decanoyl-N-methylglucamide, Triton® X-100, Triton® X-l 14, Thesit®, Isotridecypoly(ethylene
glycol ether)n, N-dodecyl--N,N-dimethyl-3-ammonio-l-propane sulfonate,
3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS):, or
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be
desirable to immobilize either BFLP0169 protein or its target molecule to facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate
automation of the assay. Binding of a test compound to BFLP0169 protein, or interaction of
BFLP0169 protein with a target molecule in the presence and absence of a candidate compound,
can be accomplished in any vessel suitable for containing the reactants. Examples of such
vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a
fusion protein can be provided that adds a domain that allows one or both of the proteins to be
bound to a matrix. For example, GST-BFLP0169 fusion proteins or GST-target fusion proteins
can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or
glutathione derivatized microtiter plates, that are then combined with the test compound or the
test compound and either the non-adsorbed target protein or BFLP0169 protein, and the mixture
is incubated under conditions conducive to complex formation (e.g., at physiological conditions
for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove
any unbound components, the matrix immobilized in the case of beads, complex determined
either directly or indirectly, for example, as described, supra. Alternatively, the complexes can
be dissociated from the matrix, and the level of BFLP0169 protein binding or activity determined
using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening
assays of the invention. For example, either the BFLP0169 protein or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin. Biotinylated BFLP0169 protein or
target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques
well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, HI), and
immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively,
antibodies reactive with BFLP0169 protein or target molecules, but which do not interfere with
58

binding of the BFLP0169 protein to its target molecule, can be derivatized to the wells of the
plate, and unbound target or BFLP0169 protein trapped in the wells by antibody corrugation.
Methods for detecting such complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes using antibodies reactive
with the BFLP0169 protein or target molecule, as well as enzyme-linked assays that rely on
detecting an enzymatic activity associated with the BFLP0169 protein or target molecule.
In another embodiment, modulators of BFLP0169 protein expression are identified in a
method wherein a cell is contacted with a candidate compound and the expression of BFLP0169
mRNA or protein in the cell is determined. The level of expression of BFLP0169 mRNA or
protein in the presence of the candidate compound is compared to the level of expression of
BFLP0169 mRNA or protein in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of BFLP0169 mRNA or protein expression
based upon this comparison. For example, when expression of BFLP0169 mRNA or protein is
greater (i.e., statistically significantly greater) in the presence of the candidate compound than in
its absence, the candidate compound is identified as a stimulator of BFLP0169 mRNA or protein
expression. Alternatively, when expression of BFLP0169 mRNA or protein is less (statistically
significantly less) in the presence of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of BFLP0169 mRNA or protein expression. The level of
BFLP0169 mRNA or protein expression in the cells can be determined by methods described
herein for detecting BFLP0169 mRNA or protein.
In yet another aspect of the invention, the BFLPO169 proteins can be used as "bait
proteins" in a two-hybrid assay or three hybrid assay, to identify other proteins that bind to or
interact with BFLPO 169 ("BFLPO 169-binding proteins" or "BFLPO 169-bp") and modulate
BFLPO 169 activity. Such BFLPO 169 -binding proteins are also likely to be involved in the
propagation of signals by the BFLPO 169 proteins as, for example, upstream or downstream
elements of the BFLPO 169 pathway.
The two-hybrid system is based on the modular nature of most transcription factors,
which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two
different DNA constructs. In one construct, the gene that codes for BFLPO 169 is fused to a gene
59

encoding the DNA binding domain of a known transcription factor {e.g., GAL-4). In the other
construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified
protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known
transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo,, forming a
BFLP0169-dependenht complex, the DNA-binding and activation domains of the transcription
factor are brought into close proximity. This proximity allows transcription of a reporter gene
(e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the
transcription factor. Expression of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to obtain the cloned gens that encodes
the protein which interacts with BFLP0169.
The invention further pertains to novel agents identified by the aforementioned screening
assays and uses thereof for treatments as described herein.
The invention will be illustrated in the following non-limiting examples.
Example 1. Expression patterns of murine BFLP0169 sequence in disease-free, lupus
nephritis simulated disease, and rapamycin-treated diseased mice
The expression of murine BFLP0169 sequences were examined in mice that developed
lupus nephritis-like symptoms in the art-recognized NZB XNZW murine model (see, e.g.,
Drake et al., Genetic analysis of the NZB contribution to lupus-like autoimmune disease in (NZB
x NZW)F1 mice,. Proc Natl Acad Sci U S A 91 -.4062-66, 1994; Finck et al., Interleukin 6
promotes murine lupus in NZB/NZWFl mice, J. Clin. Invest 94:585-91, 1994; Guglielmotti et
al., Bindarit prlongs survival and reduces renal damage of NSB/Wlupus mice. Clin. Exp.
Rheumatol. 16:149, 1998 ;Yang et al., Dietary conjugated linoleic acid protects against end
stage disease of systemic lupus erythematosus in the NZB/WF1 mouse, Immunopharmacol.
Immunotoxicol. 22:433-49,2000. Expression in diseased mice was compared to expression of
the sequences in non-diseased mice of varying ages, and in mice whose lupus nephritis-like
symptoms diminished following treatment with rapamycin or anti-B7 antibodies.
60

Mice were obtained from Jackson Laboratories at 6 to 8 weeks of age and aged on site.
Data were obtained from kidneys of mice and harvested at the indicated time point: C57BL/6
female mice at 8, and 32 weeks, Fl(NZBxNZW) female mice 12, 25, and 42 weeks, mice treated
with rapamycin at 42 and 55 weeks, mice treated with antibodies to B7.1 and B7.2 at 52 weeks.
Each group contained three mice.
Rapamycin treated mice received 5 mg/kg rapamycin subcutaneous injection 3 times per
week for 8 weeks staring at 29 weeks of age. Control mice received injections of vehicle (methyl
cellulose) on the same schedule. Effectiveness of therapy was determined by normalization of
proteinuria and kidney histology (data not shown). Gene expression analysis was preformed on
mice sacrificed at the end of the treatment course (36 weeks of age, data not shown), and at 42
weeks (6 weeks after treatment) and 55 weeks (20 weeks after treatment).
Mice treated with anti-B7 received 200ug of anti-B7.1 (1G10F9 monoclone.1) and 200u.g
of anti-B7.2 (GL1 monoclonal) by intra-peritoneal injections 3 times per week for 1wo weeks
starting at 29 weeks of age. Gene expression analysis was performed 21 weeks after treatment.
RNA isolation and hybridization to oligomicleotide arrays
Kidneys from both male and female mice were collected and snap frozen for RNA
isolation. One half each kidney was used. A longitudinal section of the left kidney and a cross
section of the right kidney was used in for each individual animal.
Snap frozen mouse kidney tissue was homogenized using homogenizer suspended in RLT
buffer plus 2ME for 30 to 45 seconds. Total RNA was prepared using the Qiagen Midi Kit
following the manufacturer's protocol. RNA was suspended in DEPC treated H2O and
quantified by OD 280.
cDNA was synthesized from 5ug of total RNA using the Superscript Kit (BRL). cDNA
was purified using phenol:cloroform:isoamyl alcohol (25:24:1) with a Phage lock gel tube
following the Phage lock protocol. Supernanant was collected and cleaned up using EtOH.
Sample was resuspended in DEPC treated H2O.
In vitro T7 polymerase driven transcription reactions for synthesis and biotin labeling of
antisense cRNA. Qiagen RNeasy spin column purification used used to purify the cRNA.
61

GeneChip hybridization mixtures contained 15ug fragmented cRNA, 0.5mg/ml acetylated BSA,
0.lmg/ml herring sperm DNA, in 1XMES buffer in a total volume of 200ul as per manufactures
instructions. Reaction mixtures were hybridized for 16hr at 45 °C to Affymetrix. Mul IKsubA
and Mul IKsubB oligonucleotide arrays. The hybridization mixtures were removed and the
arrays were washed and stained with Streptavidin R-phycoerthrin (Molecular Probes) using
GeneChip Fluidics Station 400 and scanned with a Hewlett Packard GeneArray Scanner
following manufactures instructions. Fluorescent data was collected and converted to gene
specific difference average using MicroArray Suite software.
Analysis of Oligonucleotide Array Data
An eleven member standard curve, comprised of gene fragments derived from cloned
bacterial and bacteriophage sequences were spiked into each hybridization mixture at
concentrations ranging from 0.5pM to 150pM representing RNA frequencies of approximately
3.3 to 1000 parts per million (ppm). The biotinylated standard curve fragments were synthesized
by T7-polymerase driven IVT reactions from plasmid-based templates. The spiked biotinylated
RNA fragments serve both as an internal standard to assess chip sensitivity and as standard curve
to convert measured fluorescent difference averages from individual genes into RNA frequencies
in ppm as described by Hill et al.
Gene expression frequencies from each individual mouse kidney were meiisured and the
expression data subjected to statistical analysis. Frequency values determined from individual
measurements for a given group of mice were averaged. Genes whose frequencies differed
significantly between C57B16 kidneys at 12 and 32 weeks of age were classified as changing as a
result of the normal aging process, and not due to a disease process.
Expression frequencies in young (disease-free), old (diseased), and effectively treated old
(disease-free) Fl(NZBxNZW) mice and C57BL6 control mice of oligonucleotide sequence
identified on the Affymetrix Murine 1 IK chip by the qualifier aa002653_s_at are shown. This
sequence represents an unknown mouse gene.
The results are shown in FIG. 1. Shown is a histogram showing gene expression levels in
kidneys from the indicated mice. Expression levels of BFLP0169 do not vary significantly
between C57BL/6 kidneys at 12 weeks of age and kidney at 32 weeks of age, indicating that
62

expression levels do not increase with age in kidneys of non-diseased mice. In (NXBxNZW)Fl
kidneys, the gene is expressed at normal levels prior to disease onset (12 weeks of age). As the
mice age and disease progresses, increasing expression levels are observed at 25 weeks, 36
weeks (data not shown for 36 weeks), and 42 weeks. By 55 weeks of age, the mice have died
due to kidney failure. Mice treated with rapamycin for 8 weeks with treatment starting at 29
weeks of age, remain healthy past 55 weeks of age. Kidneys of mice that have received effective '
therapy (either rapamycin therapy or anti-B7 therapy) express normal levels of BFLP0169, and
these normal levels persist in asymptomatic kidney 20 weeks after cessation of rapamycin
therapy and 15 weeks after cessation of anti-B7 therapy. The observation that expression levels
return to normal when kidney function is normal indicates that elevated levels are related to, and
diagnostic of, disease progression. Blocking the function of these genes may inhibit or retard
disease progression. Expression levels may also to used to assess and compare effectiveness of
various therapeutic interventions.
Example 2. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the L at position 78 of
the BFLP0169 sequence shown in Table 2 has been replaced by a V, which is shown in bold font.


VGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSR'iTRRGAPSSPAT
GVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRHMQR (SEQ ID NO:3)
Example 3. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the L at position 198 of
the BFLP0169 sequence shown in Table 2 has been replaced by an I, which is shown in bold
font.

Example 4. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the R at position 267 of
the BFLP0169 sequence shown in Table 2 has been replaced by a K, which is shown in bold font.

Example 7. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the L at position 553 of
the BFLPO169 sequence shown in Table 2 has been replaced by an I, which is shown in bold
font.


VNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLK
ILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLC;ELDKKKEPLTQA
VGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPAT
GVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRHMQR (SEQ ID NO:8)
Example 8. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the W at position 634
of the BFLP0169 sequence shown in Table 2 has been replaced by a F, which is shown in bold
font.

Example 9. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the N at position 749 of
the BFLP0169 sequence shown in Table 2 has been replaced by a D, which is shown in bold font.



Example 10. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the V at position 838 of
the BFLP0169 sequence shown in Table 2 has been replaced by a M, which is shown in bold
font.


A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the G at position 958 of
the BFLP0169 sequence shown in Table 2 has been replaced by a T, which is shown in bold font.

MIRKSKITSVLSFCRSSRELMTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKEL
GLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERH
PYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPS
DLLV1TKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDC
LMLTFGDIPHHAPVLLAWALLRHTLNPEETSSWRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLS
FVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLPALVSGKSTAKKV
YSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLF
TCEIEMLLHWSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTV
LAARNPAKVWTDLRHTGFLPFVAHPVSSL3QMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGST
QSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCIICSLAYTEAGQT
VINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNWSPLEQAL5IQHGAHGNNLIAV
LAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGL
IELFLNLEVKDGSDGSKEFSLTMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRD£!AMLVLRTKPKFW
ENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYWKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAE
TEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSWRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLIL
LRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIA
LFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRIJPILPTLLTTLEV
SLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLS
MSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEVJHFHLPQLMRDIQ
VNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLK
ILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQA

GVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRHMQR (SEQ ID NO:12)
Example 12. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the K at position 1084
of the BFLP0169 sequence shown in Table 2 has been replaced by a R, which is shown in bold
font.

MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPP3PSSAEKVFCANKDVASPLKEL
GLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERH
PYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPS
DLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDC
LMLTFGDIPHHAPVLLAWALLRHTLNPEETSSWRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLS
FVLTSLELHTLGNQQDIIDTACEVXADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKV
YSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPPCLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLF
TCEIEMLLHWSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTV
LAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGST
QSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQT
. VINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNWSPLEQA1SQHGAHGNNLIAV
LAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGL
IELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFW
ENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYWKGSLDQSLKDTLKKFSIEKRFAYWSGYVRSLAVHVAE
TEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSWRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLIL
LRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTBCAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIA
LFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEV
69


Example 13. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the A at position 1152
of the BFLP0169 sequence shown in Table 2 has been replaced by a S, which is shown in bold
font.

Example 14. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the I at position 1247 of
the BFLP0169 sequence shown in Table 2 has been replaced by a V, which is shown in bold font.



Example 15. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
71
A polypeptide sequence varying by one amino acid from the BFLP0169 artiino acid
sequence presented in Table 2 is shown below. For the sequence shown, the K at position 1331
of the BFLP0169 sequence shown in Table 2 has been replaced by a R, which is shown in bold
font.


A polypeptide sequence varying by one araino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the C at position 1449
of the BFLP0169 sequence shown in Table 2 has been replaced by a Y, which is shown in bold
font.

Example 17. A variant of the human BFLP0169 polypeptide sequence shown in Table .2
A polypeptide sequence varying by one amino acid from the BFLP0169 amino acid
sequence presented in Table 2 is shown below. For the sequence shown, the D at position 1542
of the BFLP0169 sequence shown in Table 2 has been replaced by a Q, which is shown in bold
font.



Example 18. A variant of the human BFLP0169 polypeptide sequence shown in Table 2
73
A polypeptide sequence varying by one amino acid from the BFLP0169 airdno acid
sequence presented in Table 2 is shown below. For the sequence shown, the F at position 1706
of the BFLP0169 sequence shown in Table 2 has been replaced by a H, which is shown in bold
font.


OTHER EMBODIMENTS
While the invention has been described in conjunction with the detailed description
thereof, the foregoing description is intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other aspects, advantages, and
modifications are within the scope of the following claims.
74

What is claimed is:
1. An isolated nucleic acid molecule encoding a polypeptide comprising an
amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:2.
2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid
molecule encodes a polypeptide that binds rapamycin.
3. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid
molecule encodes a polypeptide at least 98% identical to the amino acid sequence of SEQ
IDN0:2.
4. The nucleic acid molecule of claim 1, wherein said molecule hybridizes
under stringent conditions to a nucleic acid sequence complementary to a nucleic acid
molecule comprising nucleotides 1-1811 of SEQ ID NO: 1.
5. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid
molecule encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
6. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises nucleotides 1-1811 of SEQ ID NO:1.

7. A vector comprising the nucleic acid molecule of claim 1.
8. A cell including the vector of claim 7.
9. A substantially purified polypeptide comprising an amino acid sequence at
least 95% identical to the amino acid sequence of SEQ ID NO:2.
75

10. The polypeptide of claim 9, wherein said polypeptide binds rapamycin.
11. The polypeptide of claim 9, wherein the amino acid sequence of said
polypeptide is at least 98% identical to the amino acid sequence of SEQ ID NO:2.
12. The polypeptide of claim 9, wherein the amino acid sequence of said
polypeptide is at least 99% identical to the amino acid sequence of SEQ ID NO:2.
13. The polypeptide of claim 9, wherein the amino acid sequence of said
polypeptide comprises the amino acid sequence of SEQ ID NO:2.
14. The polypeptide of claim 9, wherein the amino acid sequence of said
polypeptide consists of the amino acid sequence of SEQ ID NO:2.
15. A fusion polypeptide comprising the polypeptide of claim 9 operably
linked to a non-BFLP0169 polypeptide.

16. The fusion polypeptide of claim 9, wherein said non-BFLP0169
polypeptide comprises at least one member selected from the group consisting of an Fc
region of an immunoglobulin molecules or a FLAG epitope, a HIS tag, and a MYC tag.
17. A polypeptide comprising a rapamycin-binding domain of the amino acid
sequence of SEQ ID NO:2.
18. A polypeptide at least 1740 amino acids in length comprising at least five
contiguous amino acids of SEQ ID NO:2, provided that said polypeptide comprises an an
amino acid sequence other than SEQ ID NO:26.
76

19. The polypeptide of claim 18, wherein said polypeptide comprises a
rapamycin-binding domain.
20. A polypeptide comprising at least five contiguous amino acids of SEQ ID
NO:20.
21. The polypeptide of claim 20, wherein said polypeptide comprises a
rapamycin-binding domain.
22. The polypeptide of claim 20, wherein said polypeptide is at least 50 amino
acids in length.
23. The polypeptide of claim 20, wherein said polypeptide is at least 100
amino acids in length.
24. The polypeptide of claim 20, wherein said polypeptide is ai least 220
amino acids in length.
25. A pharmaceutical composition comprising the polypeptide of claim 17
and a pharmaceirtically acceptable carrier.
26. A fusion polypeptide comprising a rapamycin-binding domain of a
BFLP0169 polypeptide operably linked to a non-BFLP0169 polypeptide.
27. The fusion polypeptide of claim 26, wherein said non-BFLP0169
polypeptide comprises at least one member selected from the group consisting of an Fc
region of an immunoglobulin molecules or a FLAG epitope, a HIS tag, and a MYC tag.
28. A pharmaceutical, composition comprising the fusion polypeptide of claim
26 and a pharmaceutically acceptable carrier.
77

29. An antibody that binds selectively to the polypeptide of claim 9.
30. The antibody of claim 29, wherein said antibody inhibits binding of a
BFLP0169 polypeptide to rapamycin.
31. The antibody of claim 29, wherein said antibody is a polyclonal antibody.
32. The antibody of claim 29, wherein said antibody is a monoclonal antibody.
33. The monoclonal antibody of claim 32, wherein said monoclonal antibody
is selected from the group consisting of a murine monoclonal antibody, and a humanized
monoclonal antibody.
34. A method of producing a BFLPO169 polypeptide, said method comprising
culturing a cell including the nucleic acid molecule of claim 1 under conditions allowing
for expression of a BFLPO 169 polypeptide encoded by said nucleic acid molecule.
35. A method of detecting the presence of a BFLP0169 nucleic; acid molecule
in a biological sample, the method comprising:
contacting the sample with a nucleic acid probe that binds specifically to a
BFLPO 169 nucleic acid; and
identifying the bound probe, if present,
thereby detecting the presence of BFLP0169 nucleic acid molecule in said sample.
36. A method of detecting the presence of a BFLPO 169 polypeptide in a
sample, the method comprising:
contacting the sample with a compound that selectively binds to said polypeptide
under conditions allowing for formation of a complex between said polypeptide and said
compound; and
78

detecting said complex, if present, thereby identifying said polypeptide in said
sample.
37. The method of claim 36, wherein said compound is rapamycin.
38. The method of claim 36, wherein said compound is an anti-BFLP0169
antibody.
39. A method for determining the presence of or predisposition to lupus
nephritis in a subject, the method comprising:

a) measuring the amount of a BFLPO169 nucleic acid molecule in a sample from
said subject; and
b) comparing the amount of said nucleic acid in step to the amount of the nucleic
acid present in a control sample from a subject without lupus nephritis,
wherein an increase in the ievei of said BFLP0I69 nucleic acid in step (a) as
compared to the level of the nucleic acid in the control sample indicates the presence of
or predisposition to lupus nephritis in said subject.
40. The method of claim 39, wherein said subject is a human.
41. A method for determining the presence of or predisposition to lupus
nephritis in a subject, the method comprising:
a) measuring the amount of a BFLPO 169 polypeptide in a sample from said
subject; and
b) comparing the amount of said polypeptide to the amount of the nucleic acid
present in a control sample from a subject without lupus nephritis,
wherein an increase in the level of said BFLPO 169 polypeptide as compared to the
level of the polypeptide in the control sample indicates the presence of or predisposition
to lupus nephritis in said subject.
79

42. The method of claim 41, wherein said subject is a human.
43. A method for screening for a therapeutic agent for treating an autoimmune
disorder, the method comprising:
contacting a test compound with a BFLP0169 polypeptide; and
determining if said test compound binds to said BFLP0169 polypeptide,
wherein binding of said test compound to said polypeptide indicates the test
compound is a therapeutic agent for an autoimmune disorder.
44. The method of claim 43, wherein said immune disorder is an autoimmune
disorder.
45. The method of claim 44, wherein said autoimmune disorder is lupus.
46. The method of claim 44, wherein said autoimmune disorder is lupus
lephritis.

47. The method of claim 43, wherein said BFLP0169 is provided in a cell-free
extract.
48. The method of claim 43, wherein said BFLP1069 is provided in a cell.
49. A method of treating lupus nephritis in a subject, the method comprising
administering to said subject a therapeutically effective amount of an agent that inhibits
activity of a BFLP0169 polypeptide in said subject.
50. The method of claim 49, wherein said subject is a human.
51. The method of claim 49, wherein said agent is an anti-BFLPO 169
antibody.
80

52. A pharmaceutical composition comprising an agent that inhibits activity of
a BFLP0169 polypeptide in a subject and a pharmaceutically acceptable carrier.
53. The pharmaceutical composition of claim 52, wherein said agent is an
anti-BFLP0169 antibody.
81
54. An isolated nucleic acid molecule encoding a polypeptide particularly with reference tc
sequence listing.

The present invention provides novel isolated BFI-P0169 polynucleotides and polypeptidcs encoded by the
BFLP0169 polynucleolides. Also provided are the antibodies that immunospecifically bind to a BFLP0 169 polypeptide or any
derivative (including fusion derivative), variant, mutant or fragment of the BFLP0169 polypeptide, polynucleotide or antibody.
The invention additionally provides methods in which the BFLP0169 polypeptide, polynucleotide and antibody are utilized in the
detection and treatment of a broad range of pathological states, as well as to other uses.