Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/I4201
NOVEL MOLECULES OF THE T110-RELATED PROTEIN FAMILY
AND USES THEREOF
Cross Reference To Related Applications
This application is a continuation.-in-part of
application serial number 09/102,705, filed June 22,
1998.
Bac:kQround of the Invention
The invention relates to a novel cell surface or
secreted protein and the gene encodi~rg it.
Many membrane-associated and secreted proteins,
for example, cytokines, play a vital role in the
regulation of cell growth, cell differentiation, and a
variety of specific cellular responses. A number of
medically useful proteins, including erythropoietin,
granulocyte-macrophage colony stimulating factor, human
growth hormone, and various interleukins, are secreted
proteins. Thus, an imporl~ant goal in the design and
development of new therapies is the identification and
characterization of membrane-associated and secreted
proteins and the genes which encode them.
Many membrane-associated proteins are receptors
which bind a ligand and transduce an intracellular
signal, leading to a variety of cellular responses. The
identification and characterization of such a receptor
enables one to identify both the ligands which bind to
the receptor and the intracellular molecules and signal
transduction pathways associated with the receptor,
permitting one to identify or design modulators of
receptor activity, e.g., receptor agonists or antagonists
and modulators of signal transduction.
CA 02335585 2000-12-21
WO 99/67415 PCT/(7S99/14201
- 2 -
Summary of the Invention
The presents invention is based, at least in part,
on the discovery of a gene encoding T110. T110 protein
is related to four-jointed (fj) protein of Drosophila
Melanogaster. T110 is predicted to be a member of the
type-II membrane protein superfamily. Such proteins
usually employ a transmembrane domain as the internal
signal sequence. The amino terminal end of such proteins
is normally intracellular,, and the carboxy terminal end
is normally extracellular. However, some type IT
membrane proteins are secreted from the cell while others
are initially expressed on the surface of the cell and
are subsequently processed to release a soluble fragment.
The human ~.L'110 cDNA described below (SEQ ID N0:1)
has a 1311 nucleotide open reading frame (nucleotides 131
to 1441 of SEQ ID NO:l; SEQ ID N0:3) which encodes a 437
amino acid protein (SEQ ID N0:2). Figure 8 depicts a
potential alternative translation product (SEQ ID N0:4)
for the above-described human T110 cNDA. It is possible
that this alternative translation product is not full
length. Those skilled in the art can isolate full-length
clones having additional 5' coding sequence using the
methods described below.
The mouse x'110 cDNA described below (SEQ ID N0:5)
has a 1350 nucleotide open reading frame (nucleotides 103
to 1452 of SEQ ID N0:5; SEQ ID N0:7) which encodes a 450
amino acid protein (SEQ ID N0:6). Figure 6 depicts a
potential alternative translation product (SEQ ID N0:8)
for the above-described murine T110 cDNA. It is possible
that this alternative translation product is not full
length. Those skilled in the art can isolate full-length
clones having additional 5' coding sequence using the
methods described below.
A partial rat T110 cDNA is also described below
(SEQ ID N0:9). It has a 507 nucleotide open reading
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 3 -
frame . (nucleotides 1 to 507 of . SEQ TD N0: 9} which encodes
a 169 amino acid peptide (SEQ ID N0:10). Those skilled
in the art can isolate. full-length clones having
additional 5' sequence using the methods described below.
The T110 molecules of the present invention are
useful as modulating agents in regulating a variety of
cellular processes, e.g., cell proliferation and/or cell
differentiation. .Accordingly, in one aspect, this
invention provides isolated nucleic acid molecules
encoding T110 proteins or biologically active portions
thereof, as well as nucleic acid fragments suitable, as
primers or hybridization probes far the detection of
T110-encoding nucl-eic acids.
The invention features a nucleic acid molecule
which includes a fragment of at least 400 (450, 500, 550,
600, 650, 700, 800, 900, )_000, 1100, 1200, 1300, 1400, or
1420) nucleotides of the nucleotide sequence shown in SEQ
TD NO:1 or a complement thereof; or a fragment of at
least 200 (250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 800, 900, 1000, 1110, 1200, 1300, 1400, or 1420)
nucleotides of the nucleotide sequence shown in SEQ ID
N0:3 or a complement thereof; or a fragment of at least
450 (500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200,
1300, 1400, or 145D) nucleotides of the nucleotide
sequence shown in ~SEQ ID N0:5 or SEQ ID N0:7, or a
complement thereof.
In a preferred embodiment, a T110 nucleic acid
molecule has the rnucleotide sequence shown in SEQ ID
NO:1, or SEQ ID N0:3, or SEQ ID N0:5, or SEQ ID N0:7.
Also withiru the invention is a nucleic acid
molecule which encodes a fragment of a polypeptide having
the amino acid sequence of SEQ ID N0:2 or SEQ ID N0:4, or
SEQ ID N0:6 or SEQ ID N0:8, the fragment including at
least 70 (80, 90, :100, 120, 140, 160, 180, 200, 250, 300,
350, 400, 450, or 480) contiguous amino acids of SEQ ID
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 4 -
N0:2 or SEQ ID N0:4; or the fragment including at least
150 {160, 170, 180, 200, 250, 300, 350, 400, 450, or 480)
contiguous amino acids of SEQ ID N0:6 or SEQ ID N0:8.
The invention includes a nucleic acid molecule
which encodes a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID
N0:2 or SEQ ID N0:4, or SEQ ID N0:6 or SEQ ID N0:8,
wherein the nucleic acid molecule hybridizes to a nucleic
acid molecule comprising SEQ ID NO:l or SEQ TD N0:3, or
SEQ ID N0:5 or SEQ ID N0:7 under stringent conditions.
Also within the invention is an isolated T110
protein which is encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under
stringent hybridization canditions to a nucleic acid
molecule having th~5 nucleotide sequence of SEQ ID N0:3,
SEQ ID N0:7, or SEQ ID N0:9.
Also within the invention is a polypeptide which
is a naturally occurring allelic variant of a polypeptide
that includes the <~mino acid sequence of SEQ ID N0:2 or
SEQ ID N0:4, or SEc~ ID N0:6 or SEQ ID N0:8, wherein the
polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID
N0:1 or SEQ ID N0::3, or SEQ ID N0:5 or SEQ ID N0:7 under
stringent conditions;
Another embodiment of the invention features T110
nucleic acid molecules which specifically detect T110
nucleic acid molecules. For example, in one embodiment,
a TI10 nucleic acid molecule hybridizes under stringent
conditions to a nucleic acid molecule comprising the
nucleotide sequencE~ of SEQ ID NO:1, SEQ ID N0:3, SEQ ID
N0:5, or SEQ ID N0:7, or a. complement thereof. In
another embodiment,, the T110 nucleic acid molecule is at
least 440 (450, 50c), 550, 600, 650, 700, 800, 900, 1000,
1100, 1200, 1300, :L400, or 1420) nucleotides in length
and hybridizes under stringent conditions to a nucleic
CA 02335585 2000-12-21
WO 99167415 PCT/US99/14201
- 5 -
acid molecule comb>rising the nucleotide sequence as shown
in SEQ ID N0:1 or a complement thereof; or a fragment of
at least 220 (250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or
1420) nucleotides in length and hybridizes under
stringent conditions to a nucleic acid molecule
comprising the nucleotide sequence as shown in SEQ ID
N0:3 or a complement thereof; or a fragment of at least
450 (500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200,
1300, 1400, or 1420) nucleotides in length and hybridizes
under stringent conditions to a nucleic acid molecule
comprising the nucleotide sequence as shown in SEQ ID
N0:5 or SEQ ID N0:7, or a complement thereof. In another
embodiment, the iruvention provides an isolated nucleic
acid molecule which is antisense to the coding strand of
a T110 nucleic acid.
Another aspect of the invention provides a vector,
e.g., a recombinar~t expression vector, comprising a T110
nucleic acid molecule of the invention. In another
embodiment the invention provides a host cell containing
such a vector. The invention also provides a method for
producing T110 protein by culturing, in a suitable
medium, a host cell of the invention containing a
recombinant expre~;sion vector such that a T110 protein is
produced.
Another aspect of this invention features isolated
or recombinant T11.0 proteins and polypeptides. Preferred
T110 proteins and polypeptides possess at least one
biological activity possessed by naturally occurring
human T110, e.g., modulation of cellular proliferation.
In one embodiment, an isolated T110 protein has an
extracellular domain and lacks both a transmembrane and a
cytoplasmic domain. In another embodiment, an isolated
T110 protein is soluble under physiological conditions.
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 6 -
The T110 proteins of the present invention, or
biologically active portions thereof, can be operably
linked to a non-T~_10 polypeptide (e. g., heterologous
amino acid sequences) to form T110 fusion proteins. The
invention further features antibodies that specifically
bind T110 proteins;, such as monoclonal or polyclonal
antibodies. In addition, the T110 proteins or
biologically active portions thereof can be incorporated
into pharmaceutical compositions, which optionally
include pharmaceutically acceptable carriers.
In another aspect, the present invention provides
a method far detecaing the presence of T110 activity or
expression in a biological sample by contacting the
biological sample with an agent capable of detecting an
indicator of T110 activity such that the presence of T110
activity is detected in the biological sample.
In another aspect, the invention provides a method
for modulating T11.0 activity comprising contacting a cell
with an agent that. modulates (inhibits ar stimulates)
T110 activity or expression such that T110 activity or
expression in the cell is modulated. In one embodiment,
the agent is an antibody that specifically binds to T110
protein. In another embodiment, the agent modulates
expression of T110 by modulating transcription of a T110
gene, splicing of a T110 mRNA, or translation of a T110
mRNA. Tn yet another embodiment, the agent is a nucleic
acid molecule having a nucleotide sequence that is
antisense to the coding strand of the T110 mRNA or the
T110 gene.
In one embodiment, the methods of the present
invention are used. to treat a subject having a disorder
characterized by aberrant T110 protein activity or
nucleic acid expression by administering an agent which
is a T110 modulator to the subject. In one embodiment,
the T110 modulator is a T110 protein. In another
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
_ 7 _
embodiment the T11.0 modulator is a T110 nucleic acid
molecule. In other embodiments, the T110 modulator is a
peptide, peptidomi.metic, or other small molecule. In a
preferred embodim~:nt, the disorder characterized by
aberrant T110 protein or nucleic acid expression is
neaplasia, inappropriate angiogenesis, or inappropriate
tissue regeneration after injury.
The presents invention also provides a diagnostic
assay for identifying the presence or absence of a
genetic lesion or mutation characterized by at least one
of: (i) aberrant modification or mutation (including
partial or complete deletion or amplification) of a gene
encoding a T110 protein; (ii} mis-regulation of a gene
encoding a T110 protein; and (iii} aberrant post-
translational modification of a T110 protein, wherein a
wild-type form of the gene encodes a protein with a T110
activity.
In another aspect, the invention provides a
method for identifying a compound that binds to or
modulates the activity of a T110 protein. In general,
such methods entail measuring a biological activity of a
T110 protein in the presence and absence of a test
compound and identifying those compounds which alter the
activity of the T110 protein.
The invent:~on also features methods for
identifying a compound which modulates the expression of
T110 by measuring the expression of T110 in the presence
and absence of a compound.
A plasmid containing DNA encoding human T110 and a
plasmid containing DNA encoding murine T110 were
deposited with the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Virginia,
20110-2209, on June 22, 1998, and have been assigned ATCC
Accession Nos. and , respectively. The
deposits were made according to the terms of the Budapest
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
_ g _
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure. The
plasmid containing human DNA was deposited in E. coli
(strain designation Epfthb 110d), which contains a human
T110 DNA in the plasmid vector pZLl. The plasmid
containing murine DNA was also deposited in E: coZi
{strain designation Epftmb 110g), which contains a murine
T110 DNA in the plasmid vector pZLI. The deposits were
made merely as a convenience for those of skill in the
art and are not an admission that deposits are required
under 35 U.S.C. ~ 112.
The invention includes a nucleic acid molecule
that contains the nucleotide sequence of the cDNA having
ATCC Accession No. , the coding sequence of that
cDNA (i.e., the cD:L~A having ATCC Accession No. ),
or complements thereof. Similarly, the invention
includes a nucleic acid molecule that contains the
nucleotide sequence of the cDNA having ATCC Accession No.
the coding sequence of that cDNA (i.e., the cDNA
having ATCC Accession No. ), or complements
thereof .
The invent9.on includes polypeptides encoded by the
coding sequence of the nucleic acid molecules described
above, i.e., seque:nce contained within the nucleic acid
molecules deposited with t:he ATCC and assigned ATCC
Accession Nos. and ~, and biologically
active fragments thereof. Moreover, those of ordinary
skill in the art will recognize that many, if not all, of
the methods described herein can be practiced with the
nucleic acid molecvules (or complements or fragments
thereof} deposited with the ATCC, as described above,
and/or the polypeptides {or fragments thereof) encoded by
those molecules, just as they can be practiced as
described herein by reference to a given SEQ ID No.
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/I420I
- 9 -
Other feat;ores, and advantages of the invention
will be apparent from the following detailed description
and claims.
Brief Description of the Drawings
Figure 1 dlepicts the cDNA sequence (SEQ ID N0:1}
and predicted amino acid sequence (SEQ ID N0:2) of human
T110. The open reading frame of SEQ ID NO:1 extends from
nucleotide 131 to nucleotide 1441 (SEQ ID N0:3).
Figure 2 is a hydropathy plot of human T110. The
location of the predicted transmembrane (TM), and
extracellular (OU'.C) domains are indicated as are the
position of cyste:i.nes (cys; vertical bars) and potential
glycosylation sites (Ngly; vertical bars). Relative
hydrophobicity is shown above the dotted line, and
relative hydrophi:Licity is shown below the dotted line.
Figure 3 depicts the cDNA sequence (SEQ ID N0:5)
and predicted amino acid sequence (SEQ ID NO:&) of mouse
T110. The open reading frame of SEQ ID N0:5 extends from
nucleotide 103 to nucleotide 1452 (SEQ ID N0:7).
Figure 4 is a hydrapathy plot of mouse T110. The
location of the predicted, transmembrane (TM), and
extracellular (OU'T) domains are indicated as are the
position of cyste9.nes (cys; vertical bars) and potential
glycosylatin site~r (Ngly; vertical bars). Relative
hydrophobicity is shown above the dotted line, and
relative hydrophi7.icity is shown below the dotted line.
Figure 5A .depicts the partial cDNA sequence of rat
T110 (SEQ ID N0:9).
Figure 5B depicts the predicted amino acid
sequence (SEQ ID TTO:10) of rat T110. The coding region
of SEQ ID NO:10 Extends from nucleotide 1 to nucleotide
507 of SEQ ID NO:S~.
Figure 6 depicts the cDNA sequence (SEQ ID NO:1)
and predicted amino acid sequence (SEQ ID N0:4) of a
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- to -
potential alternative human T110 translation product.
The open reading fvrame extends from nucleotide 2 to 1411
of SEQ ID NO:1 (SE;Q ID N0:12).
Figure 7 i;s a hydropathy plot of a potential
alternative human T110 translation product. The location
of the predicted t.ransmembrane (TM), and extracellular
(OUT) domains are indicated as are the position of
cysteines (cys; vertical bars) and potential
glycosylation sites (Ngly; vertical bars). Relative
hydrophobicity is shown above the dotted line, and
relative hydrophilicity is shown below the dotted line.
Figure 8 despicts the cDNA sequence (SEQ ID N0:5)
and predicted amino acid sequence (SEQ ID N0:8) of a
potential alternative murine T110 translation product.
The open reading frame extends from nucleotide 1 to 1452
of SEQ ID N0:5 (SEQ ID NO:13).
Figure 9 i~~ a hydropathy plot of a potential
alternative murine T110 translation product. The
location of the predicted transmembrane (TM), and
extracellular (OUT) domains are indicated as are the
position of cysteines (cys; vertical bars) and potential
glycosylation sites (Ngly; vertical bars). Relative
hydrophobicity is shown above the dotted line, and
relative hydrophilicity is shown below the dotted line.
Figure 10 depicts the sequence alignment of
D. melanogaster four jointed protein (SEQ ID N0:11) with
human T110 protein (SEQ ID N0:2).
Figure 11 9.s a plot showing predicted structural
features of a potential alternative human T110 protein.
Detailed Description of the Invention
The present: invention is based on the discovery of
a cDNA molecule encoding human T110, a member of the
type--II membrane protein superfamily. A nucleotide
sequence encoding .a human T110 protein is shown in Figure
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 11 -
1 (SEQ ID N0:1; SF~Q ID,N0:3 includes the open reading
frame only). A predicted amino acid sequence of T110
protein is also shown in Figure 1 (SEQ ID NO: 2). This
protein includes a predicted signal peptide of about 28
amino acids (from amino acid 1 to about amino acid 28 of
SEQ ID N0:2). The: predicted mature protein extends from
about amino acid 29 to amino acid 437 of SEQ ID N0:2 (SEQ
ID N0:14).
The human 'T110 cDNA of Figure 1 (SEQ ID N0:1),
which is approximately 2401 nucleotides long including
untranslated regions, encodes a protein amino acid having
a molecular weight. of approximately 48 kDa (excluding
post-translational. modifications).
Human T110 protein and D. me3anogaster four-
jointed (fj) protean share many primary features. They
are proteins of similar size and both contain a single
predicted hydrophc>bic region near the N-terminus that may
be a transmembrane: domain rather than a signal sequence.
Thus, the hydrophobic region from amino acids 1-28 (or 7-
30) might be a tramsmembrane domain that acts as an
internal signal sequence. Each protein contain two pairs
of conserved cyste:ine residues, one pair near the center
of the molecule (cyslsl and cysl~e) , the other pair near the
C-terminus of the molecule (cys3ss and cys42,) . Regions of
highest identity between the two proteins surround the
two pairs of cyste:ines in the extracellular domains.
Each protein also contains putative N-glycosylation
sites, two of which are in approximately the same
position, i.e., beaween the two pairs of cysteines (amino
acid residuess 24f. to 251 and amino acid residues 277 to
280). A sequence alignment of human T110 protein and
D. melanogaster fj protein is depicted in Figure 6. In
this alignment, human T110 protein and D. melanogaster fj
protein display about 30% identity and about 36%
similarity.
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 12 -
An approxivmately 2.4 kb human T110 mRNA transcript
is expressed at the highest level in brain, heart,
placenta, and pancreas. Low levels of this transcript
have been observed in liver, skeletal muscle, and kidney.
No detectable message is seen in lung. Embryonic
expression is seen in week 8-9 fetus and week 20 liver
and spleen mixed tissues. Embryonic expression is also
observed in neuronal tissue.
Human T110 is one member of a family of molecules
(the "T110 family"~ having certain conserved structural
and functional fe~~tures. The present invention provides
detailed description of various members of the "T110
family", e.g., human T110, mouse T110, and rat T110. The
term "family" when referring to the protein and nucleic
acid molecules of the invention is intended to mean two
or more proteins c>r nucleic acid molecules having a
common structural domain and having sufficient amino acid
or nucleotide sequence identity as defined herein. Such
family members can be naturally occurring and can be from
either the same ox' different species. For example, a
family can contain a first protein of human origin and a
homologue of that protein of murine origin, as well as a
second, distinct ~>rotein of human origin and a murine
homologue of that protein. Members of a family may also
have common functional characteristics.
Preferred 'T110 po7.ypeptides of the present
invention have an amino acid sequence sufficiently
identical to the consensus amino acid sequence of human
T110 protein. As used herein, the term "sufficiently
identical" refers to a first amino acid or nucleotide
sequence which contains a sufficient or minimum number of
identical or equivalent (e. g., an amino acid residue
which has a similar side chain) amino acid residues or
nucleotides to a ~>econd amino acid ar nucleotide sequence
such that the fir:~t and second amino acid or nucleotide
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 13 -
sequences have a common structural domain and/or common
functional activity. For example, amino acid ar
nucleotide sequences which contain a common structural
domain having about 65% identity, preferably 75%
identity, more preferably 85%, 95%, or 98% identity are
defined herein as sufficiently identical.
As used interchangeably herein a "T110 activity",
"biological activity of T110" or "functional activity of
T110", refers to an activity exerted by a T110 protein,
polypeptide or nucleic acid molecule on a T110 responsive
cell as determined in vivo, or an vitro, according to
standard techniques. A T110 activity can be a direct
activity, such as an association with or an enzymatic
activity on a sect>nd protein or an indirect activity,
such as a cellular signaling activity mediated by
interaction of the T110 protein with a second protein.
In a preferred embodiment, a T110 activity includes at
least one or more of the following activities: (i) the
ability to interacts with proteins in the T110 signalling
pathway (ii) the ability to interact with a T110 ligand
or receptor (iii} the ability to interact with an
intracellular target protein; and (iv) the ability to
interact with proteins involved in cellular proliferation
or differentiatior.~.
Accordingly, another embodiment of the invention
features isolated T110 proteins and polypeptides having a
T110 activity.
Various aspects of the invention are described in
further detail in the follawing subsections.
I. Isalated Nucleic Acid Molecules
One aspect of the invention pertains to isolated
nucleic acid malecules that encode T110 proteins or
biologically active portions thereof, as well as nucleic
acid molecules sufficient for use as hybridization probes
CA 02335585 2000-12-21
WO 99167415 PCTIUS99/14201
- 14 -
to identify T110-esncoding~ nucleic acids (e.g., T110 mRNA)
and fragments for use as PCR primers for the
amplification or mutation of T110 nucleic acid molecules.
As used herein, the term "nucleic acid molecule" is
intended to include DNA molecules (e. g., cDNA or genomic
DNA) and RNA molecules (e.g., mRNA) and analogs of the
DNA ar RNA generated using nucleotide analogs. The
nucleic acid molecule can be single-stranded or double-
stranded, but preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which
is separated from other nucleic acid molecules which are
present in the natural source of the nucleic acid.
Preferably, an "ieoolated" nucleic acid is free of
sequences (preferably protein encoding sequences) which
naturally flank the nucleic acid (i.e., sequences located
at the 5' and 3' emds 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 T110 nucleic acid molecule can contain Iess than
about 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 o~enomic :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
substantially free: 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 N0:1, SEQ ID N0:3, SEQ ID N0:5, SEQ ID
N0:7, ar a complement of any of these nucleotide
sequences, can be isolated using standard molecular
biology techniques and the sequence information provided
herein. Using all or a portion of the nucleic acid
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 15 -
sequences of SEQ ID N0:1, SEQ ID N0:3, SEQ ID N0:5, or
SEQ ID N0:7 as a hybridization probe, T110 nucleic acid
molecules can be isolated. using standard hybridization
and cloning techn»ques (e.g., as described in Sambrook et
al . , eds . , Mo.~ ecu~Car Cl oning: A Labora tory Manual . 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989).
A nucleic acid of the invention can be amplified
using cDNA, mRNA c>r genomic DNA as a template and
appropriate oligonucleotide primers according to standard
PCR amplification techniques. The nucleic acid so
amplified can be c:Ioned into an appropriate vector and
characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to T110 nucleotide
sequences can be prepared by standard synthetic
techniques, e.g., using an automated DNA synthesizer.
In another preferred embodiment, an isolated
nucleic acid molecule of the invention comprises a
nucleic acid molecule which is a complement of the
nucleotide sequence shown in SEQ ID NO:1, SEQ ID N0:3,
SEQ ID N0:5, SEQ I:D N0:7, or a portian thereof. A
nucleic acid molecule which is complementary to a given
nucleotide sequence is one which is sufficiently
complementary to the given nucleotide sequence that it
can hybridize to the given nucleotide sequence thereby
forming a stable aluplex.
Moreover, ithe nucleic acid molecule of the
invention can comprise only a portion of a nucleic acid
sequence encoding T110, for example, a fragment which can
be used as a probe: or primer or a fragment encoding a
biologically active portion of T110. The nucleotide
sequence determined from the cloning of the human T110
gene allows for the generation of probes and primers
designed for use in identifying and/or cloning T110
homologues in other cell types, e.g., fram other tissues,
CA 02335585 2000-12-21
WO 99/67415 PCT/US99l14201
- 16 -
as well as T110 homologues from other mammals. The
probe/prirner typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises
a region of nucleotide sequence that hybridizes under
stringent conditions to at least about 12, preferably
about 25, more preferably about 50, 75, 100, 125, 150,
175, 200, 250, 300, 350 or. 400 consecutive nucleotides of
the sense or anti-sense sequence of SEQ ID NO:1, SEQ ID
N0:3, SEQ ID N0:5, SEQ ID N0:7 or of a naturally
occurring mutant of SEQ ID N0:1, SEQ ID N0:3, SEQ ID
N0:5, SEQ ID N0:7.
Probes basE:d on the human T110 nucleotide sequence
can be used to detect transcripts or genomic sequences
encoding the same ~or identical proteins. The probe
comprises a label .group attached thereto, e.g., 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 far identifying cells or tissues
which mis-express .a T110 protein, such as by measuring a
level of a T110-encoding nucleic acid in a sample of
cells from a subject, e.g., detecting T110 mRNA levels or
determining whether a genomic T110 gene has been mutated
or deleted.
A nucleic acid fragment encoding a "biologically
active portion of 'T110" can be prepared by isolating a
portion of SEQ ID NO:l, SEQ ID N0:3, SEQ ID N0:5, SEQ ID
N0:7 which encodes a polypeptide having a T110 biological
activity, expressing the encoded portion of T110 protein
(e. g., by recombinant expression in vztro) and assessing
the activity of the encoded portion of T110.
The invention further encompasses nucleic acid
molecules that differ from the nucleotide sequence of SEQ
ID NO:1, SEQ ID N0:3, SEQ ID N0:5, or SEQ ID N0:7 due to
degeneracy of the !genetic code and thus encode the same
T110 protein as that encoded by the nucleotide sequence
CA 02335585 2000-12-21
WO 99/67415 PCT/EJS99/14201
- 17 -
shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5, or SEQ ID
N0:7.
In addition to the human T110 nucleotide sequence
shown in SEQ ID NO:1 and SEQ ID N0:3, it will be
appreciated by those skilled in the art that DNA sequence
polymorphisms that: lead to changes in the amino acid
sequences of T110 may exist within a population (e. g.,
the human populati.on). Such genetic polymorphism in the
T110 gene may.exi~;t among individuals within a population
due to natural allelic variation. An allele is one of a
group of genes which occur alternatively at a given
genetic locus. As', used herein, the terms "gene" and
"recombinant gene°° refer to nucleic acid molecules
comprising an open, reading frame encoding a T110 protein,
preferably a mammalian T110 protein. Such natural
allelic variations can typically result in 1-5~ variance
in the nucleotide sequence of the T110 gene. Alternative
alleles can be identified by sequencing the gene of
interest in a number of different individuals. This can
be readily accomplished using hybridization probes
recognizing T110 sequences to identify the same genetic
locus in a variety of individuals. Any and all such
nucleotide variations and resulting amino acid
polymorphisms in T110 that are the result of natural
allelic variation and that do not alter the functional
activity of T110 are intended t~ be within the scope of
the invention.
Moreover, nucleic acid molecules encoding T110
proteins from other species (T110 homologues), which have
a nucleotide sequence which differs from that of a human
T110, are intended to be within the scope of the
invention. Nucleic acid molecules corresponding to
natural allelic variants and homologues of the T110 cDNA
of the invention can be isolated based on their identity
to the human T110 :nucleic acids disclosed herein using
CA 02335585 2000-12-21
WO 99167415 PCT/US99/14201
- 1$ -
the human cDNAs, o:r a portion thereof, as a hybridization
probe according to standard hybridization techniques
under stringent hybridization conditions. For example, a
soluble human T110 cDNA can be isolated based on its
identity to human membrane-bound T110. Likewise, a cDNA
encoding a membrane-bound form of human T110 can be
isolated based on its identity to soluble human T110.
Accordingly, in another embodiment, an isolated
nucleic acid molecule of the invention is at least 440
(450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100,
1200, 1300, 1400, or 1420) nucleotides in length and
hybridizes under stringent conditions to a nucleic acid
molecule having the nucleotide sequence as shown in SEQ
ID N0:1 or a complement thereof; or the isolated nucleic
acid molecule is at least 220 (250, 300, 350, 400, 450,
500, 550, &00, 650, 700, 800, 900, 1000, 1100, 1200,
1300, 1400, or 1420) nucleotides in length and hybridizes
under stringent conditions to a nucleic acid molecule
having the nucleotide sequence as shown in SEQ ID N0:3 or
a complement thereof; or the isolated nucleic acid
molecule is at Least 450 (500, 550, 600, 650, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, or 1450) nucleotides
in length and hybridizes under stringent conditions to a
nucleic acid molecule comprising the nucleotide sequence
as shown in SEQ ID~ N0:5 or SEQ ID N0:7, or a complement
thereof .
As used herein, the term "hybridizes under
stringent conditions" is intended to desoribe conditions
for hybridization and washing under which nucleotide
sequences at least 60% (65%, 70%, preferably 75%, 85%, or
95%) identical to each other typically remain hybridized
to each other. SU.Ch stringent conditions are known to
those skilled in the art and can be found in Current
Protocols in Molec.~ular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. A preferred, non-limiting example
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 19 -
of stringent hybr:idization conditions are hybridization
in 6X sodium chloa:ide/sodium citrate (SSC) at about 45°C,
followed by one or more washes in 0.2 X SSC, 0.1% SDS at
50-65°C. Preferably, an isolated nucleic acid molecule
of the invention that hybridizes under stringent
conditions to the sequence of SEQ ID NO:1, SEQ ID N0:3,
SEQ ID N0:5 or SEQ ID N0:7 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).
In addition to naturally-occurring allelic
variants of the T1.10 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, SEQ ID N0:3, SEQ ID
N0:5 or SEQ ID N0:7, thereby leading to changes in the
amino acid sequence of the encoded T120 protein, without
altering the functional ability of the T110 protein. For
example, one can make nucleotide substitutions leading to
amino acid substitutions at "non-essential" amino acid
residues. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence
of T110 (e.g., the sequence of SEQ ID N0:2 or SEQ ID
N0:5) without altering the biological activity, whereas
an "essential" amino acid residue is required for
biological activity. For example, amino acid residues
that are conserved among the T110 proteins of various
species are predicted to be particularly unamenable to
alteration.
For example:, preferred T110 proteins of the
present invention ~~ontain at least two pairs of conserved
cysteines in the e:Ktracellular domain. Such conserved
amino acids are legs likely to be amenable to mutation.
Other amino acid residues, however, (e.g., those that are
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 20 -
not conserved or only semi-conserved among T110 of
various species) may not be essential for activity and
thus are likely to be amenable to alteration.
In another example, preferred T110 proteins of the
present invention contain residues that are identical
between human T110 and mouse T110. Such conserved amino
acids between human T110 and mouse T110 are likely to be
structurally or functionally significant. Accordingly,
it is preferable t~o preserve these conserved residues
unless one wishes to decrease protein stability or
activity.
Accordingly, another aspect of the invention
pertains to nuclei~~ acid molecules encoding T110 proteins
that contain changes in amino acid residues that are not
essential for activity. Such T110 proteins differ in
amino acid sequence from SEQ ID N0:2, 4, & or 8 yet
retain biological activity. In one embodiment, the
isolated nucleic acid molecule includes a nucleotide
sequence encoding a protein that includes an amino acid
sequence that is at least about 65% identical, 75%, 85%,
95%, or 98% identical to the amino acid sequence of SEQ
TD N0:2, 4, 6, or .B.
An isolatedl nucleic acid molecule encoding a T110
protein having a s~squence which differs from that of SEQ
ID N0:2, 4, 6, or 8 can be created by introducing one or
more nucleotide substitutions, additions or deletions
into the nucleotide sequence of SEQ ID N0:1, SEQ ID N0:3,
SEQ ID N0:5, or SEl2 ID N0:7 such that one or more amino
acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced 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
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 21 -
the amino acid re;>idue,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, i.soleucine, 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 T110
is preferably replaced with another amino acid residue
from the same side chain family. Alternatively,
mutations can be introduced randomly along all or part of
a T110 coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened
for T110 biological activity to identify mutants that
retain activity. Following mutagenesis, the encoded
protein can be expressed recombinantly and the activity
of the protein can be determined.
In a preferred embodiment, a mutant T110 protein
can be assayed for: (1) the ability to form
protein: protein interactions with proteins in a T110
signalling pathway; (2) the ability to bind a T110 ligand
or receptor (3) the ability to bind to an intracellular
target protein; or (~) the ability to interact with a
protein involved i:n cellular proliferation or
differentiation. In yet another preferred embodiment, a
mutant T210 can be assayed for the ability to modulate
cellular proliferation or cellular differentiation.
The present: invention encompasses antisense
nucleic acid molecules, i.e., molecules which are
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 22 -
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. Accordingly, an antisense nucleic acid can
hydrogen bond to a sense nucleic acid. The antisense
nucleic acid can be complementary to an entire T110
coding strand, or to only a portion thereof, e.g., all or
part of the protein coding region (or apen reading
frame). An antise:nse nucleic acid molecule can be
antisense to a nor.~coding region of the coding strand of a
nucleotide sequence encoding T110. The noncoding regions
("5' and 3' untranslated regions") are the 5' and 3'
sequences which flank the coding region and are not
translated into amino acids.
Given the coding strand sequences encoding T110
disclosed herein (e. g., SEQ ID NO:l or S~Q ID N0:3),
antisense nucleic acids of the invention can be designed
according to the rules of Watson and Crick base pairing.
The antisense nucleic acid molecule can be complementary
to the entire coding region of T110 mRNA, but more
preferably is an c~ligonucleotide which is antisense to
only a portion of the coding or noncoding region of T110
mRNA. For example., the antisense oligonucleotide can be
complementary to the region surrounding the translation
start site of T110 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 and 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 si:ability of the malecules or to
increase the physical stability of the duplex formed
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 23 -
between the antisc~nse and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted
nucleotides can bE~ used. Examples of modified
nucleotides which 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-carboxymethyl-arninomethyl-2-thiouridine, 5-
carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine~, inosine, N&-iso-pentenyladenine, 1-
methylguanine, 1-naethylinosine, 2,2-di-methylguanine, 2-
methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-
methylcytosine, NEB-adenine, 7-methylguanine,
5-methylaminometh~~luracil, 5-methoxyaminomethyl-2-
thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxy-
methyluracil, 5-meahoxyuracil, 2-methyl-thio-N6-
isopentenyladenine, uracil-5-oxyacetic acid (v),
wybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyl-uracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-
amino-3-N-2-carbox:ypropyl} uracil, (acp3)w, and 2,6-
diaminopurine. Alternatively, the antisense nucleic acid
can be produced biologically using an expression vector
25~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 T110
protein to thereby inhibit expression of the protein,
e.g., by inhibiting transcription and/or translation.
CA 02335585 2000-12-21
WO 99/67415 PCT/LJS99/14201
- 24 -
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 which
binds to DNA duplexes, through specific interactions in
the major groove of the 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 antise:nse nucleic acid molecules to peptides
or antibodies which 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 the
antisense molecules, vector constructs in which the
antisense nucleic acid molecule is placed under the
control of a stron~3 pol II or pol III promoter are
preferred.
An antisens~,e nucleic acid molecule of the
invention can be an a-anomeric nucleic acid molecule. An
a-anomeric nucleic acid molecule forms specific double-
stranded hybrids with complementary RNA in which,
contrary to the usual ~i-units, the strands run parallel
to each other (Gau:Ltier et al. (1987) Nuc.Ieic Acids. Res.
15:6625-6641). The antisense nucleic acid molecule can
also comprise a 2'~-o-methylribonucleotide (moue et al.
(1987) Nucleic Acids Res. 15:6131-6148) or a chimeric
RNA-DNA analogue (:Cnoue et al. (1987) FEBS Lett. 215:327-
330) .
The invention also encompasses ribozymes.
Ribozymes are cata:Lytic RNA molecules with ribonuclease
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 25 -
activity which are' capable of cleaving a single-stranded
nucleic acid, such as an mRNA, to which they have a
complementary reguon. Thus, ribozymes (e. g., hammerhead
ribozymes (described in Haselhoff and Gerlach (1988)
Nature 334:585-597L}) can be used to catalytically cleave
T110 mRNA transcr~.pts to thereby inhibit translation of
T110 mRNA. A ribozyme having specificity far a T110-
encoding nucleic acid can be designed based upon the
nucleotide sequence of a T110 cDNA disclosed herein
(e.g., SEQ ID NO:J., SEQ ID N0:3). For example, a
derivative of a Te~trahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the
active site is complementary to the nucleotide sequence
to be cleaved in a T110-encoding mRNA. See, e.g., Cech
et al. U.S. Patent: No. 4,987,071; and Cech et al. U.S.
Patent No. 5,116,i'42. Alternatively, T110 mRNA can be
used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See,
e.g., Bartel and ~~zostak (1993} Science 261:1411-1418.
The invention alsa encompasses nucleic acid
molecules which farm triple helical structures. For
example, T110 gene expression can be inhibited by
targeting nucleotide sequences complementary to the
regulatory region of the T110 (e. g., the T110 promoter
and/ar enhancers) to form triple helical structures that
prevent transcription of the T110 gene in target cells.
See generally, Helene {1991) Anticancer Drug Des.
6(6):559-84; Helene (1992) Ann. N.Y. Acad. Sci. &60:27-
36; and Maher (1992) Bioassays 14(12):807-15.
In preferred embodiments, the nucleic acid
molecules of the invention can be modified at the base
moiety, sugar moieay or phosphate backbone to improve,
e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate
backbone of the nucleic acids can be modified to generate
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 26 -
peptide nucleic acids (see Hyrup et al. (1996) Bioorganic
& Medicinal Chemistry 4(1): 5-23). 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 as described in Hyrup et al.
(1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93: 14670-675.
PNAs of T1L0 can be used in therapeutic and
diagnostic applications. For example, PNAs can be used
as antisense or antigene agents far sequence-specific
modulation of gene expression by, e.g., inducing
transcription or translation arrest or inhibiting
replication. PNAs of T110 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., S1 nucleases (Hyrup (1996) supra; or as
probes or primers for DNA sequence and hybridization
(Hyrup (1996) supra; Perry-O'Keefe et al. (1996) Proc.
Nati . Acad. Sci . D~SA 93 : 14670-675) .
In another embodiment, PNAs of T110 can be
modified, e.g., to enhance their stability or cellular
uptake, by attaching Iipophilic 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 T110 can be
generated which may combine the advantageous properties
of PNA and DNA. Such chimeras allow DNA recognition
enzymes, e.g., RNA.se H and DNA polymerases, to interact
CA 02335585 2000-12-21
WO 99/67415 PCTIUS99/14201
- 27 -
with the DNA portion while the.PNA portion would provide
high binding affinity and specificity. PNA-DNA chimeras
can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds
between the nucleobases, and orientation (Hyrup (7.996)
supra). The synthesis of PNA-DNA chimeras can be
performed as described in Hyrup (1996) supra and Finn
et al. (1996) Nuc~.eic Acids Research 24 (17) :3357-63. For
example, a DNA chain can be synthesized on a solid
support using standard phosphoramidite coupling chemistry
and modified nucleoside analogs. Compounds such as 5'-
(4-meth-oxytrityl)amino-5'-deoxy-thymidine
phosphoramidite can be used as a link between the PNA and
the 5' end of DNA (Mag et al. (1989) Nucleic Acid Res.
17:5973-88). PNA monomers are then coupled in a stepwise
manner to produce a chirneric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al. (1996) Nucleic
Acids Research 24(17):3357-63). Alternatively, chimeric
molecules can be synthesized with a 5' DNA segment and a
3' PNA segment (Peterser et al. (1975) B.ioorganic Med.
Chem. Lett. 5:1119-11124).
In other ennbodiments, the oligonucleotide may
include other appended groups such as peptides (e.g., for
targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (see,.
e.g., Letsinger et al. {1989) Proc. Nat.l. Acad. Sci. USA
86:6553--6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or
the blood-brain barrier (see, e.g., PCT Publication No.
WO 89/10134). In addition, oligonucleotides can be
modified with hybridization-triggered cleavage agents
(see, e.g., Krol et al. (1988) B.io/Techniques 6:958-976)
or intercalating agents (see, e.g., Zon (1988) Pharm.
Res. 5:539-549). To this end, the oligonucleotide may be
conjugated to another molecule, e.g., a peptide,
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 28 -
hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
II. Isolated T110 Proteins and Anti-T110 Antibodies
One aspect of the invention pertains to isolated
T110 proteins, an~L biologically active portions thereof,
as well as polypeptide fragments suitable for use as
immunogens to rais~~e anti-T110 antibodies. In one
embodiment, native: T110 proteins can be isolated from
cells or tissue sources by an appropriate purification
scheme using standard protein purification techniques.
In anather embodiment, T110 proteins are produced by
recombinant DNA techniques. Alternative to recombinant
expression, a T110 protein or polypeptide can be
synthesized chemically using standard peptide synthesis
techniques.
An "isolatESd" or "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 T110 protein is
derived, or substantially free of chemical precursors or
other chemicals when chemically synthesized. The
language "substantially free of cellular material"
includes preparations of T110 protein in which the
protein is separated from cellular components of the
cells from which it is isolated or recombinantly
produced. Thus, T110 protein that is substantially free
of cellular material includes preparations of T110
protein having less than about 30%, 20%, 10%, or 5% (by
dry weight) of non-T110 protein (also referred to herein
as a "contaminating protein"). When the T110 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%, 10%, or 5% of the volume of the protein
-- CA 02335585 2000-12-21
WO 99/67415 PCT/US99/I42UI
- 29 -
preparation. When. T110 protein is produced by chemical
synthesis, it is preferably substantially free of
chemical precursors or other chemicals, i.e., it is
separated from chemical precursors or other chemicals
which are involved. in the synthesis of the protein,
Accordingly such preparations of T110 protein have less
than about 30%, 20~, 10°s, 5~ (by dry weight} of chemical
precursors or non-T110 chemicals.
Biological~Ly active portions of a T110 protein
include peptides comprising amino acid sequences
sufficiently identical to or derived from the amino acid
sequence of the T110 protein (e. g., the amino acid
sequence shown in SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6,
or SEQ ID N0:8}, which include fewer amino acids than the
full length T110 proteins,. and exhibit at least one
activity of a T110 protein. Typically, biologically
active portions comprise a domain or motif with at least
one activity of the T110 protein. A biologically active
portion of a T110 ;protein can be a polypeptide which is,
for example, 10, 25, 50, 100 or more amino acids in
length. Preferred biologically active polypeptides
include one or more identa.fied T110 structural domains,
e.g., the extracellular domain (SEQ ID N0:4 and SEQ ID
N0:8}.
w 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 T110
protein. Preferred T110 protein has the amino acid
sequence shown of ;SEQ ID N0:2 or SEQ ID N0:6. Other
useful T110 proteins are substantially identical to SEQ
ID N0:2 or SEQ ID ~nT0:6 and retain the functional activity
of the protein of ;SEQ ID N0:2 or SEQ ID N0:6 yet differ
in amino acid sequence due to natural allelic variation
or mutagenesis. A~~cordingly, a useful TliO protein is a
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 30 -
protein which includes, an amino acid sequence at least
about 45%, prefera.biy 55%, 65%, 75%, 85%, 95%, or 99%
identical to the amino acid sequence of SEQ ID N0:2 or
SEQ ID N0:6 and retains the functional activity of the
T110 proteins of SEQ ID N0:2. In other instances, the
T110 protein is a protein having an amino acid sequence
55%, 65%, 75%, 85%, 95%, or 98% identical to the T110
extraceilular domain (SEQ ID N0:4 or SEQ ID N0:8). In a
preferred embodiment, the T110 protein retains a
functional activity of the T110 protein of SEQ ID N0:2 or
SEQ ID NO:6.
To determine the percent identity of two amino
acid sequences or of two nucleic acids, the sequences are
aligned for optimal comparison purposes (e.g., gaps can
be introduced in the sequence of a first amino acid or
nucleic acid sequence for optimal alignment with a second
amino or nucleic acid sequence). The amino acid residues
or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position
in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in
the second sequence, then the molecules are identical at
that position. Th~s percent identity between the two
sequences is a function of the number of identical
positions shared b;y the sequences (i.e., % identity =
of identical posit:ions/total ## of overlapping positions x
100). Preferably, the two sequences are the same length.
The determination of percent homology between two
sequences can be accomplished using a mathematical
algorithm. A preferred, non-limiting example of a
mathematical algorithm utilized for the comparison of two
sequences is the algorithm of Karlin and Altschul (1990)
Pros. Nat'1 Acad. ,Sci. USA 87:2264-2268, modified as in
Karlin and Altschu:l {1993) Proc. Nat'I Acad. Sci. USA
90:5873-5877. Suclz an algorithm is incorporated into the
CA 02335585 2000-12-21
WO 99167415 PCTIUS99/14201
- 31 -
NBLAST, and XBLAST ~?rograms of Altschul, et al. (1990)
J. Mol. Biol. 215:403-410. BLAST nucleotide searches can
be performed with the NBLAST program, score = 100,
wordlength = 12 to obtain nucleotide sequences homologous
to T110 nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST
program, score = 50, wordlength = 3 to obtain amino acid
sequences homologous to T110 protein molecules of the
invention. To obtain gapped alignments for comparison
purposes, Gapped B1C~AST can be utilized as described in
Altschul et al., (:1997) Nucleic Acids Res. 25:3389-3402.
When utilizing BLAST and Gapped BLAST pragrams, the
default parameters of the respective programs (e. g.,
XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov. Another preferred, non-
limiting example o:f a mathematical algorithm utilized for
the comparison of sequences is the algorithm of Myers and
Miller, CABIOS (19;89). Such an algorithm is incorporated
into the ALIGN program (version 2.0) which is part of the
GCG sequence alignment software package. When utilizing
the ALIGN program :for comparing amino acid sequences, a
PAM120 weight residue table, a gap length penalty of 12,
and a gap penalty of 4 can be used.
The percent. identity between two sequences can be
determined using techniques similar to those described
above, with or without allowing gaps. In calculating
percent identity, .only exact matches are counted.
The invention also provides T110 chimeric or
fusion proteins. :As used herein, a T110 "chimeric
protein" or "fusio:n protein" comprises a T110 polypeptide
operably linked to a non-T110 polypeptide. A "T110
polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to T110, whereas a "non-T110
polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not
CA 02335585 2000-12-21
WO 99!67415 PCT/US99/14201
- 32 -
substantially identical to the.T110 protein, e.g., a
protein which is different from the T110 protein and
which is derived from the same or a different organism.
Within a T110 fusion protein the T110 polypeptide can
correspond to all or a portion of a T110 protein,
preferably at least one biologically active portion of a
T110 protein. Within the fusion protein, the term
"operably linked" is intended to indicate that the T110
polypeptide and the non-T110 polypeptide are fused in-
frame to each other. The non-T110 polypeptide can be
fused to the N-terminus or C-terminus of the T110
polypeptide.
One useful fusion protein is a GST-T110 fusion
protein in which the T110 sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins
can facilitate the purification of recombinant T110.
In yet another embodiment, the fusion protein is
an T110-immunoglobulin fusion protein in which all or
part of T110 is fused to sequences derived from a member
of the immunoglobulin protein family. The T110-
immunoglobulin fusion proteins of the invention can be
incorporated into pharmaceutical compositions and
administered to a subject to inhibit an interaction
between a T110 ligand and a T110 protein on the surface
of a cell, to thereby suppress T110-mediated signal
transduction in vivo. The T110-immunoglobulin fusion
proteins can be used to affect the bioavailability of a
T110 cognate ligan.d. Inhibition of the T110 ligand/T110
interaction may be useful therapeutically for both the
treatment of proliferative and differentiative disorders,
as well as modulating (e. g. promoting or inhibiting) cell
survival. Mareove.r, the T110-immunoglobulin fusion
proteins of the invention can be used as immunogens to
produce anti-T110 antibodies in a subject, to purify T110
ligands and in screening assays to identify molecules
CA 02335585 2000-12-21
WO 99167415 PCT/US99I14201
- 33 -
which_inhibit the :interaction of T110 with a T110 ligand.
Preferably, a T110 chimeric or fusion protein of
the invention is 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, fox
example 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. Tn 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 which
give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be
annealed and reamplified to generate a chimeric gene
sequence (see, e.g., Current Protoco3s in Molecular
Biology, Ausubel et al. eds., John Wiley & Sons: 1992).
Moreover, many expression vectors are commercially
available that already encode a fusion moiety (e.g., a
GST polypeptide). An T110-encoding nucleic acid can be
cloned into such an expression vector such that the
fusion moiety is linked in-frame to the T110 protein.
The present: invention also pertains to variants of
the T110 proteins {i.e., proteins having a sequence which
differs from that of a naturally occurring T110) which
function as either T110 agonists {mimetics) or as T110
antagonists. Variants of the T110 protein can be
generated by mutagenesis, e.g., discrete point mutation
or truncation of the T110 protein. An agonist of the
T110 protein can retain substantially the same, ar a
subset, of the biological activities of the naturally
CA 02335585 2000-12-21
WO 99/67415 PCT/IJS99114201
- 34 -
occurring form of the T120 protein. An antagonist of the
T110 protein can inhibit one or more of the activities of
the naturally occurring form of the T110 protein by, for
example, competitively binding to a downstream or
upstream member of a cellular signaling cascade which
includes the T110 protein. Thus, specific biological
effects can be elicited by treatment with a variant of
limited function. Treatment of a subject with a variant
having a subset of the biological activities of the
naturally occurring form of the protein can have fewer
side effects in a subject relative to treatment with the
naturally occurring form of the T110 proteins.
Variants oi: the T110 protein which function as
either T110 agonists (mimetics) or as T110 antagonists
can be identified by screening combinatorial libraries of
mutants, e.g., truncation mutants, of the T110 protein
for T110 protein agonist or antagonist activity. In one
embodiment, a variegated library of T110 variants is
generated by combinatorial mutagenesis at the nucleic
acid level and is encoded by a variegated gene library.
A variegated library of T110 variants can be produced by,
for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that
a degenerate set of potential T110 sequences is
expressible as individual polypeptides, or alternatively;
as a set of larger fusion proteins (e. g., for phage
display) containing the set of T110 sequences therein.
There are a variety of methods which can be used to
produce libraries of potential T110 variants from a
degenerate oligonucleotide sequence. Chemical synthesis
of a degenerate gene sequence can be performed in an
automatic DNA synthesizer, and the synthetic gene then
ligated into an appropriate expression vector. Use of a
degenerate set of genes allows for the provision, in one
mixture, of all of the sequences encoding the desired set
CA 02335585 2000-12-21
WO 99/67415 PCT/US99114201
- 35 -
of potential T110 ,3equences. Methods for synthesizing
degenerate oligonucleotides are known in the art (see,
e.g., Narang (1983;1 Tetrahedron 39:3; Itakura et al.
(1984) Anna. Rev. :Biochem. 53:323; Itakura et al. (1984)
Science 198:1056; :Ike et a1. (1983) Nucleic Acid Res.
11:477) .
In addition, libraries of fragments of the T110
protein coding sequence can be used to generate a
variegated population of T110 fragments for screening and
subsequent selection of variants of a T110 protein. In
one embodiment, a :Library of coding sequence fragments
can be generated by treating a double stranded PCR
fragment of a T110 coding sequence with a nuclease under
conditions wherein nicking occurs only about once per
molecule, denaturing the double stranded DNA, renaturing
the DNA to form double stranded DNA which can include
sense/antisense pairs from different nicked products,
' removing single stranded portions from reformed duplexes
by treatment with ;31 nuclease, and ligating the resulting
20~ fragment library into an expression vector. By this
method, an expression library can be derived which
encodes. N-terminal and internal fragments of various
sizes of the T110 protein.
Several techniques are known in the art for
screening gene products of combinatorial libraries made
by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property.
Such techniques arcs adaptable for rapid screening of the
gene libraries genf:rated by the combinatorial mutagenesis
of T110 proteins. The most widely used techniques, which
are amenable to high through-put analysis, for screening
large gene libraris=s typically include cloning the gene
library into replicable expression vectors, transforming
appropriate cells with the resulting library of vectors,
and expressing the combinatorial genes under conditions
CA 02335585 2000-12-21
WO 99167415 PCTIUS99114201
- 36 -
in which detection of a desired activity facilitates
isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a
technique which enlaances the frequency of functional
mutants in the libraries, can be used in combination with
the screening assays to identify T110 variants {Arkin and
Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;
Delgrave et al. (1993) Protein Engineering 6(3):327-331).
An isolated T110 protein, or a portion or fragment
thereof, can be used as an immunogen to generate
antibodies that bind T110 using standard techniques for
polyclonal and monoclonal antibody preparation. The
full-length T110 protein can be used or, alternatively,
the invention provides antigenic peptide fragments of
T110 far use as immunogens. The antigenic peptide of
T110 comprises at least 8 (preferably 10, 15, 20, or 30)
amino acid residues of the amino acid sequence shown in
SEQ ID N0:2 or SEQ ID NO:~ and encompasses an epitope of
T110 such that an antibody raised against the peptide
forms a specific immune complex with T110.
Preferred Espitopes encompassed by the antigenic
peptide are regions of T110 that are located on the
surface of the protein, e.g., hydrophilic regions. Other
important criteria. include a preference for a terminal
sequence, high antigenic index (e.g., as predicted by the
Jameson-Wolf algorithm), ease of peptide synthesis (e. g.,
avoidance of proli.nes), and high surface probability
(e. g., as predicted by the Emini algorithm).
A T110 immunogen typically is used to prepare
antibodies by immunizing a suitable subject, (e. g.,
rabbit, goat, mou:~e or other mammal) with the immunogen.
An appropriate imrnunogenic preparation can contain, for
example, recombinantly expressed T110 protein or a
chemically synthe;~ized T110 polypeptide. The preparation
can further include an adjuvant, such as Freund's
CA 02335585 2000-12-21
WO 9916'7415 PCT/US99/14201
37 -
complete or incomplete adjuvant, or similar
immunostimulatory agent. immunization of a suitable
subject with an immunogenic T110 preparation induces a
polyclonal anti-T110 antibody response.
Accordingly, another aspect of the invention
pertains to anti-T110 antibodies. The term "antibody" as
used herein refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen
binding site which specifically binds an antigen, such as
T110. A molecule which specifically binds to T110 is a
molecule which binds T110, but does not substantially
bind other molecules in a sample, e.g., a biological
sample, which naturally contains T110., Examples of
immunologically active portions of immunoglobulin
molecules include F'(ab) and F(ab')2 fragments which can be
generated by treating the antibody with an enzyme such as
pepsin. The invention provides polyclonal and monoclonal
antibodies that bind T110. The term "monoclonal
antibody" or "monoclonal antibody composition", as used
herein, refers to a. population of antibody molecules that
contain only one species of an antigen binding site
capable of immunore:acting with a particular epitope of
T110. A monoclonal antibody composition thus typically
displays a single binding affinity for a particular T110
protein with which it immunoreacts.
Polyclonal anti-T110 antibodies can be prepared as
described above by immunizing a suitable subject with a
T110 immunogen. The anti-'T110 antibody titer in the
immunized subject can be monitored over time by standard
techniques, such aa; with an enzyme linked immunosorbent
assay (ELISA) using immobilized T110. If desired, the
antibody molecules directed against T110 can be isolated
from the mammal (e. g., from the blood) and further
purified by well-known techniques, such as protein A
CA 02335585 2000-12-21
WO 99/67415 ' PCT/US99/14241
- 38 -
chromatography to obtain the IgG fraction. At an
appropriate time after immunization, e.g., when the anti-
T110 antibody titers are highest, antibody-producing
cells can be obtained from the subject and used to
prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by
Kohler and Milstein (1975) Nature 256:495-497, the human
B cell hybridoma technique (Kozbor et a1. (1983) Immunol
Today 4:72), the EBV-hybridoma technique (Cole et a1.
(1985), Monoc3onal .Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96) or t:rioma techniques. The
technology for producing various antibodies and
monoclonal antibody hybridomas is well known (see
generally Current Protocols in Immunology (1994) Coligan
et al. (eds.) John Wiley & Sons, Inc., New York, NY).
Briefly, an immortal cell line (typically a myeloma) is
fused to lymphocytes (typically splenocytes) from a
mammal immunized with a T110 immunogen as described
above, and the culture supernatants of the resulting
hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds T110.
Any of the many well known protocols used for
fusing lymphocytes and immortalized cell lines can be
applied for the purpose of generating an anti-T110
monoclonal antibody (see, e.g., Current Protocols in
Immunology, supra; Galfre et al. (1977} Nature 266;55052;
R.H. Kenneth, in Mo~noclona.~ Antibodies: A New Dimension
In BiologzcaZ Analyses, Plenum Publishing Corp., New
York, New York (1980); and Lerner (1981) Yale .T. BioL.
Med., 54:387-402. Moreover, the ordinarily skilled
worker will appreciate that there are many variations of
such methods which also would be useful. Typically, the
immortal cell line (e. g., a myeloma cell line) is derived
from the same mammalian species as the lymphocytes. For
example, murine hybridomas can be made by fusing
CA 02335585 2000-12-21
WO 99167415 PCT/US99/14201
._ 3 g -
lymphocytes from a mouse immunized with an immunogenic
preparation of the present invention with an immortalized
mouse cell line, e.g., a myeloma cell line that is
sensitive to culture medium containing hypoxanthine,
aminopterin and thymidine ("HAT medium"). Any of a
number of myeloma cell lines can be used as a fusion
partner according t.o standard techniques, e.g., the P3-
NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines.
These myeloma line; are available from ATCC. Typically,
HAT-sensitive mouse: myeloma cells are fused to mouse
splenocytes using polyethylene glycol ("PEG"). Hybridoma
cells resulting frc>m the fusion are then selected using
HAT medium, which kills unfused and unproductively fused
myeloma cells (unfused splenocytes die after several days
because they are not transformed). Hybridoma cells
producing a monoclc>nal antibody of the invention are
detected by screening the hybridoma culture supernatants
for antibodies that: bind T110, e.g., using a standard
ELISA assay.
Alternative to preparing monoclonal antibody-
secreting hybridomas, a monoclonal anti-T110 antibody can
be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e. g., an antibody
phage display library) with T110 to thereby isolate
immunoglobulin libx-ary members that bind T110. Kits for
generating and screening phage display libraries are
commercially available (e. g., the Pharmacies Recombinant
Phage Antibody System, Catalog No. 27-9400-01; and the
St ratagene SurfZAPT"" Phage Display Ki t, Catalog No .
240612). Additionally, examples of methods and reagents
particularly amenable for use in generating and screening
antibody display library can be found in, for example,
U.S. Patent No. 5,:?23,409; PCT Publication No. WO
92/18619; PCT PublLcation No. W0 91/17271; PCT
Publication No. WO 92/20791; PCT Publication No. WO
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 40 -
92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO
92/09690; PCT Publication No. WO 90/02809; Fuchs et al.
(1991) Bio/Technolc>gy 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomasr 3:81-85; Huse et al. (1989) Science
246:1275-1281; Grif:fiths et al. (1993) EMBO J 12:725-734.
Additionally, recombinant anti-T110 antibodies,
such as chimeric and humanized monoclonal antibodies,
comprising both human and non-human portions, which can
be made using standard recombinant DNA techniques, are
within the scope of: the invention. Such chimeric and
humanized monoclonal antibodies can be produced by
recombinant DNA tec~hn.iques known in the art, for example
using methods described in PCT Publication No. WO
87/02671; European Patent Application 184,187; European
Patent Application 171,496; European Patent Application
173,494; PCT Publication No. WO 86/01533; U.S. Patent No.
4,816,567; European Patent Application 125,023; Better et
al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc.
Nail. Acad. Sci. U4>A 84:3439-3443; Liu et al. (1987) J.
Immunol. 139:3521-3526; Sun et al. (1987) Proc. Nat./.
Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc.
Res. 47:999-1005; Vilood et al. (1985) Nature 314:446-449;
and Shaw et al. (1988) J. Nail. Cancer Inst. 80:1553-
1559); Morrison, (1.985) Science 229:1202-1207; Oi et al.
(1986) Bio/Tech.nic~,tes 4:214; U.S. Patent 5,225,539; Jones
et al.'(1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
Completely :human antibodies are particularly
desirable for therapeutic treatment of human patients.
Such antibodies can be produced using transgenic mice
which are incapablE: of expressing endogenous
immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. The
CA 02335585 2000-12-21
WO 99/67415 PCT/US99114201
-- 41 _
transgenic mice are immunized in the normal fashion with
a selected antigen, e.g., all or a portion of T110.
Monoclonal antibodies directed against the antigen can be
obtained using conventional hybridoma technology. The
human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation,
and subsequently undergo class switching and somatic
mutation. Thus, using such a technique, it is possible
to produce therapeutically useful IgG, IgA and IgE
antibodies. For an: overview of this technology for
producing human antibodies, see Lonberg and Huszar (1995,
Int: Rev. Immunol. 13:65-93). For a detailed discussion
of this technology for producing human antibodies and
human monoclonal antibodies and protocols for producing
such antibodies, sE:e, e.g., U.S. Patent 5,625,126; U.S.
Patent 5,633,425; Lf.S. Patent 5,569,825; U.S. Patent
5,661,016; and U.S. Patent 5,545,806. In addition,
companies such as Abgenix, Inc. (Freemont, CA), can be
engaged to provide human antibodies directed against a
selected antigen using technology similar to that
described above.
Completely human antibodies which recognize a
selected epitope can be generated using a technique
referred to as "guided selection." In this approach a
selected non-human monoclonal antibody, e.g., a murine
antibody, is used t:o guide the selection of a completely
human antibody recognizing the same epitope.
First, a non-human monoclonal antibody which binds
a selected antigen (epitope), e.g., an antibody which
inhibits T110 activity, is identified. The heavy chain
and the light chain of the non-human antibody are cloned
and used to create phage display Fab fragments. For
example, the heavy chain gene can be cloned into a
plasmid vector so that the heavy chain can be secreted
from bacteria. The=_ light chain gene can be cloned into a
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 42 -
phage coat protein gene so that the light chain can be
expressed on the surface of phage. A repertoire (random
collection) of human light: chains fused to phage is used
to infect the bac~.~~ria which express the non-human heavy
chain. The resulting progeny phage display hybrid
antibodies (human :Light chain/non-human heavy chain)
The selected antigen is used in a panning screen to
select phage which bind the selected antigen. Several
rounds of selection may be required to identify such
phage. Next, human light chain genes are isolated from
the selected phage which bind the selected antigen.
These selected human light chain genes are then used to
guide the selection of human heavy chain genes as
follows. The selected human light chain genes are
inserted into vectors for expression by bacteria.
Bacteria expressin~~ the selected human light chains are
infected with a repertoire of human heavy chains fused to
phage. The resulting progeny phage display human
antibodies (human :light chain/human heavy chain).
Next, the ;elected antigen is used in a panning
screen to select p;~hage which bind the selected antigen.
The phage selected in this step display completely human
antibody which recognize the same epitope recognized by
the original selected, non-human monoclonal antibody.
The genes encoding both the heavy and light chains are
readily isolated a:nd can be further manipulated for
production of human antibody. This technology is
described by Jespe:rs et al. {1994, Biotechnology
12:899-903).
An anti-T17.0 antibody (e. g., monoclonal antibody)
can be used to isolate T110 by standard techniques, such
as affinity chromatography or immunoprecipitation. An
anti-T110 antibody can facilitate the purification of
natural T110 from cells and of recombinantly produced
T110 expressed in host cells. Moreover, an anti-T110
CA 02335585 2000-12-21
WO 99!67415 PCT/US99/14201
- 43 -
antibody can be used to detect .T1.10 protein (e.g. , in a
cellular lysate or cell supernatant) in order to evaluate
the abundance and pattern of expression of the T110
protein. Anti-T110 antibodies can be used diagnostically
to monitor protein levels in tissue as part of a clinical
testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be
facilitated by coupling the antibody to a detectable
substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, (3-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of
suitable fluorescent materials include umbelliferone,
fluorescein, fluor<sscein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material
includes luminol; examples of bioluminescent materials
include luciferase, luciferin, and aequorin, and examples
of suitable radioa~~tive material include 1251, 1311, ass or
3H .
IIL. Recombinant !Expression Vectors and Host Cells
Another aspect of the invention pertains to
vectors, preferably expression vectors, containing a
nucleic acid encoding T1~.0 (or a portion 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
CA 02335585 2000-12-21
WO 99167415 PCT/fJS99/14201
g _
additional DNA segments, can be ligated into the viral
genome. Certain v~:ctors 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 epi.somal mammalian vectors). Other
vectors (e.g., non-~episomal mammalian vectors) are
integrated into they genome of a host cell upon
introduction into t:he host cell, and thereby are
replicated along with the host genome. Moreover, certain
vectors, expression vectors, are capable of directing the
expression of geneec to which they are operably linked.
In general, expres~~ion vectors of utility in recombinant
DNA techniques are often in the form of plasmids
(vectors). However, the invention is intended to include
such other forms of' expression vectors, such as viral
vectors (e. g., replication defective retroviruses,
adenoviruses and acleno-associated viruses), which serve
equivalent functions.
The recombinant expression vectors of the
invention comprise a nucleic acid of the invention in a
form suitable for expression of the nucleic acid in a
host cell, which mE:ans that the recombinant expression
vectors include onE: or more regulatory sequences,
selected on the basis of the host cells to be used for
expression; which a.s operably linked to the nucleic acid
sequence to be expressed. 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 which 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., pol.yadenylation signals). Such
CA 02335585 2000-12-21
WO 99/67415 PCT1US99/14201
-~45--
regulatory sequences are described, for example, in
Goeddel; Gene Expression Technology: Met.hads in
Enzymology 185; Academic Press, San Diego, CA (1990).
Regulatory sequences include those which direct
constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of
the nucleotide sequence only in certain host cells (e. g.,
tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design
of the expression vector can depend on such factors as
the choice of the host cell to be transformed, the level
of expression of protein desired, etc. 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., T110 proteins, mutant forms of
T110, fusion proteins, etC.).
The recombinant expression vectors of the
invention can be designed for expression of T110 in
prokaryotic or eukaryotic cells, e.g., bacterial cells
such as E. coli, insect cells (using baculovirus
expression vectors), yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel,
Gene Expression Technology: Methods in Enzymo3.ogy 185,
Academic Press, San Diego, CA (1990). Alternatively, the
recombinant expression vector can be transcribed and
translated in vitro, for example using T7 promoter
regulatory sequences and T'7 polymerase.
Expression of proteins in prokaryotes is most
often carried out in E. colt with vectors containing
constitutive or ind.ucible promoters directing the
expression of either fusion or non-fusion proteins:
Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the
recombinant protein.. Such fusion vectors typically serve
CA 02335585 2000-12-21
WO 99167415 PCTIUS99/14201
- 46 -
three purposes: 1) to increase expression of recombinant
protein; 2} to increase the solubility of the recombinant
protein; and 3) to aid in the purification of the
recombinant protein by acting as a ligand in affinity
purification. Often, in fusion expression vectors, a
proteolytic cleava<3e site is introduced at the junction
of the fusion moiety and t:he recombinant protein to-
enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion
protein. Such enzymes, and their cognate recognition
sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia
Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),
pMAL {New England :Biolabs, Beverly, MA) and pRITS
(Pharmacia, Piscat;away, NJ) which fuse glutathione
S-transferase {GST), maltose E binding protein, or
protein A, respectively, to the target recombinant
protein.
Examples of: suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., (1988)
Gene 69:301-315) a:nd pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, California (1990) 60-89).
Target gene expression from the pTrc vector relies on
host RNA polymerase transcription from a hybrid trp-lac
fusion promoter. Target gene expression from the pET 11d
vector relies on transcription from a T7 gnl0-lac fusion
promoter mediated by a coexpressed viral RNA polymerase
(T7 gn1). This viral polymerase is supplied by host
strains BL21(DE3) or HMS174(DE3} from a resident
prophage harboring a T7 gn1 gene under the
transcriptional control of: the lacUV5 promoter.
One strategy to maximize recombinant protein
expression in E. co.Zi is to express the protein in a host
bacteria with an impaired capacity to proteolytically
CA 02335585 2000-12-21
WO 99167415 PCT/US99114201
- 47 -
cleave. the recombinant .protein ~ (Gottesman, Gene
Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, California (1990) 119-128).
Another strategy i:~ to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector
so that the individual colons for each amino acid are
those preferential:Ly utilized in E. coli (Wada et al.
(1992) Nucleic Acids Res. 20:2111-2118). Such alteration
of nucleic acid sequences of the invention can be
accomplished by st<~ndard DNA synthesis techniques.
In another embodiment, the T110 expression vector
is a yeast expression vector. Examples of vectors for
expression in yeast S. cerivisae include pYepSecl
(Baldari et al. (1!x87) EMBO J. 6:229-234) , pMFa (.Kurjan
Z5 and Herskowitz, (1982) Ce.Y.l 30:933-943), pJRY88 (Schultz
et al. (1987) Gene 54:113-123), pYES2 (In~ritrogen
Corporation, San Diego, CA), and picZ (InVitrogen Corp,
San Diego, CA) .
Alternatively, T110 can be expressed in insect
cells using baculovirus expression vectors. Baculovirus
vectors available :for expression of proteins in cultured
insect cells (e. g., Sf 9 cells) include the pAc series
(Smith et al. (1983) Mol. Cel.I Biol. 3:2156-2165) and the
pVL series (Lucklow and Summers (1989) Virology 170:31-
39) .
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 pCDMB {Seed (1987) Nature
329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-
195). 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
CA 02335585 2000-12-21
WO 99/67415 PCT/US99114201
- 48 -
expression systems for both prokaryotic and eukaryoti.c
cells see chapters 16 and 17 of Sambrook et al. (supra}.
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; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters {Calame and
Eaton (1988) Adv. Iinmurrol. 43:235-275) , in particular
promoters of T cell receptors (Winoto and Baltimore
{1989) EMBO J. 8:729-733) and immunoglobulins {Banerji et
al. (1983) Cell 33:729-740; Queen and Baltimore {1983)
Cell 33:741-748), neuron-specific promoters (e.g., the
neurofilament promoter; Byrne and Ruddle (1989) Proc.
Natl . Acad. Sci . US'A 86 : 54'73-5477) , pancreas-specific
promoters (Edlund et al. (1985) Science 230:912-916), and
mammary gland-specific promoters (e. g., milk whey
promoter; U.B. Pate:nt No. 4,873,316 and European
Application Publication No. 264;166). Developmentally-
regulated promoter: are also encompassed, for example the
murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the cx-fetoprotein promoter (Campes and
Tilghman (1989) Genies Dev. 3:537-546) .
The invention furtrier provides a recombinant
expression vector comprising a DNA molecule of the
invention cloned into the expression vector in an
antisense orientat~.on. That is, the DNA molecule is
operably linked to a regulatory sequence in a manner
which allows for e~:pression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to T110
mRNA. Regulatory sequences aperably linked to a nucleic
acid cloned in the antisense orientation can be chosen
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
49 -
which 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 which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The
antisense expression vector can be in the form of a
recombinant plasmicl, phagemid or attenuated virus in
which antisense nucleic acids are produced under the
control of a high efficiency regulatory region, the
activity of which c;an be determined by the cell type into
which the vector i~; introduced. For a discussion of the
regulation of gene expression using antisense genes see
Weintraub et al . (F2eviews - Trends in Genetics, Vol. 1 (1)
1986) .
Another aspect of the invention pertains to host
cells into which a recombinant expression vector of the
invention has been intraduced. 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 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, T110 protein can be expressed in
bacterial cells such as E. ca.l.i, insect cells, yeast or
mammalian cells (such as Chinese hamster ovary cells
(CHO) or COS cells). Other suitable host cells are known
to those skilled ire the art.
Vector DNA can be introduced into prokaryotic or
eukaryotic cells vLa conventional transformation or
transfection techn_Lques. As used herein, the terms
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 5a -
"transformation" and "transfection" are intended to refer
to a variety of arty-recognized techniques for introducing
foreign nucleic acid (e. g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-
y precipitation, DEA~~-dextran-mediated transfection,
Iipofection, or ele~ctroporation. Suitable methods for
transforming or transfecti.ng host cells can be found in
Sambrook, et al. (aupra), and other laboratory manuals.
For stable transfection of mammalian cells, it is
known that, depend:Lng upon the expression vector and
transfection techn:Lque used, only a small fraction of
cells may integrate the foreign DNA into their genome.
In order to identi.Ey and select these integrants, a gene
that encodes a selcsctable marker (e.g., for resistance to
~5 antibiotics) is generally introduced into the host cells
along with the genes of interest. Preferred selectable
markers include those which confer resistance to drugs,
such as 6418, hygromycin and methotrexate. Nucleic acid
encoding a selectable marker can be introduced into a
host cell on the same vector as that encoding T110 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 ce:Lls 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) T110 protein.
Accordingly, the invention further provides methods for
producing T110 pro?tein 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 T110 has been
introduced) in a suitable medium such that T110 protein
is produced. In another embodiment, the method further
CA 02335585 2000-12-21
WO 99/67415 PCTfUS99/I4201
- 51 -
comprises isolating T11.0 from the medium or the host
cell.
The host ce:Lls of the invention can also be used
to produce nonhuman transgenic animals. For example, in
one embodiment, a host cell of the invention is a
fertilized oocyte or an embryonic stem cell into which
T110-coding sequences have been introduced. Such host
cells can then be used to create non-human transgenic
animals in which exogenous T110 sequences have been
ZO introduced into their genome or homologous recombinant
animals in which endogenous T110 sequences have been
altered. Such animals are useful for studying the
function and/or activity of T110 and for identifying
and/or evaluating modulators of T110 activity. As used
herein, a "transger.~ic animal" is a non-human animal,
preferably a mammal., more preferably a rodent such as a
rat or mouse, in wriich one or more of the cells of the
animal includes a t;ransgene. Other examples of
transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A
transgene is exogenous DNA which is integrated into the
genome of a cell fz-om which a transgenic animal develops
and which remains in the genome of the mature animal,
thereby directing t:he expression of an encoded gene
product in one or snore cell types or tissues of the
transgenic animal. As used herein, an "homologous
recombinant animal" is a non-human animal, preferably a
mammal, more preferably a mouse, in which an endogenous
T110 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 T110-encoding nucleic acid into
the male pronuclei of a fertilized oocyte, e.g., by
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 52 -
microinjection, ret:roviral infection, and allowing the
oocyte to develop Ln a pseudopregnant female foster
animal. The T110 <:DNA sequence e.g., that of (SEQ ID
N0:1, SEQ ID N0:3, SEQ ID N0:5, and SEQ ID N0:7) can be
introduced as a transgene into the genome of a non-human
animal. Alternatively, a nonhuman homologue of the human
T110 gene, such as a mouse T110 gene can be used as a
transgene. A nonhuman homologue of the human T110 can be
isolated based on hybridization to the human T110 cDNA.
Intronic sequences and polyadenylation signals can also
be included in the transgene to increase the efficiency
of expression of the transgene. A tissue-specific
regulatory sequences) can be operably linked to the T110
transgene to direct: expression of T110 protein to
Z5 particular cells. Methods for generating transgenic
animals via embryo manipulation and microinjection,
particularly anima7Ls such as mice, have become
conventional in the: art and are described, for example,
in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent
No. 4, 873, 191 and _Ln Hogan, Manipulating 'the Mouse
Embryo, (Cold Sprung Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986). Similar methods are used for
production of other transgenic animals. A transgenic
founder animal can be identified based upon the presence
of the T110 transgE:ne in its genome and/or expression of
T110 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 T110 can
further be bred to other transgenic animals carrying
other transgenes.
To create an homologous recombinant animal, a
vector is prepared which contains at least a portion of a
T110 gene (e.g., a human or a non-human homolag of the
T110 gene, e.g., a murine T110 gene) into which a
CA 02335585 2000-12-21
WO 99/67415 PCT/US9911420I
._ 5 3
deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the T110 gene.
In a preferred embc>diment, the vector is designed such
that, upon homologous recombination, the endogenous T110
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 homologc>us recombination, the endogenous T110
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 T110 protein}. Tn the homologous
recombination vector, the altered portion of the T110
gene is flanked at its 5' and 3' ends by additional
nucleic acid of thE~ T110 gene to allow for homologous
recombination to occur between the exogenous T110 gene
carried by the vector and an endogenous T110 gene in an
embryonic stem cell.. The additional flanking T110
nucleic acid is of sufficient length for successful
homologous recombination with the endogenous gene.
Typically, several kilobases of flanking DNA (both at the
5' and 3' ends) arE: included in the vector (see, e.g.,
Thomas and Capecchi (1987) Ce31 51:503 for a description
of homologous recombination vectors). The vector is
introduced into an embryonic stem cell line (e.g.,-by
electroporation) acrd cells in which the introduced T110
gene has homologau~>ly recombined with the endogenous T110
gene are selected ;see, e.g., Li et al. (1992) Cell
69:915). The selecaed cells are then injected into a
blastocyst of an animal (e. g., a mouse) to form
aggregation chimeras (see, e.g., Bradley in
Teratocarcinomas and Embryonic Stem CeIIs: A Practical
Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-
152). A chimeric Embryo can then be implanted into a
suitable pseudoprec~nant female faster animal and the
CA 02335585 2000-12-21
WO 99/67415 PCT/US99l14201
- 54 -
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. Methods for constructing
homologous recombination vectors and homologous
recombinant animals are described further in Bradley
(1991) Current Opinion in Bio/Technology 2:823-829 and in
PCT Publication Nos. WO 90/11354, WO 91/01140, WO
92/0968, and WO 93/04169.
In another embodiment, transgenic non-human
animals can be produced which contain selected systems
which allow for regulated expression of the transgene.
One example of such a system is the cre/IoxP recombinase
system of bacteriophage P1. For a description of the
cre/loxP recombinase system, see, e.g., Lakso et al.
(1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another
example of a recombinase system is the FLP recombinase
system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355. Tf a cre/loxP recombinase
system is used to regulate expression of the transgene,
animals containing transgenes encoding bath the Cre
recombinase and a selected protein are required. Such
animals can be provided through the construction of
~~doublee 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 t:he non-human transgenic animals
described herein can also be produced according to the
methods described in Wilmut et al. (1997) Nature 385:810-
813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.
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
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 55 -
then be fused, e.g., throu.gh 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 fosi:.er animal will be a clone of the
animal from which i~he cell, e.g., the somatic cell, is
isolated.
IV. Pharmaceutica:L Compositions
The T110 nucleic acid molecules, T110 proteins,
and anti-T110 antibodies (also referred to herein as
"active compounds") of the invention can be incorporated
into pharmaceutica:L compositions suitable for
administration. Such compositions typically comprise the
nucleic acid molecule, protein, or antibody and a
pharmaceutically a~~ceptable carrier. As used herein the
language "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. The use
of such media and .agents for pharmaceutically active
substances is well known in the arty 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
(topical), transmucosal, and rectal administration.
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 56 -
Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the
following camponent:s: a sterile diluent such as water for
injection, saline solution, fixed ails, polyethylene
glycols, glycerine,. propylene glycol ar other synthetic
solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or
sodium bisulfate; c:helating agents such as
ethylenediamine-tet:raacetic acid; buffers such as
acetates, citrates or phosphates and agents for the
adjustment of tonic:ity such as sodium chloride or
dextrose. pH can be adjusted with acids ar bases, such
as hydrochloric ac~.d or sodium hydroxide. The parenteral
preparation can be enclosed in ampoules, disposable
syringes or multip7_e dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injection
include sterile aqueous solutions (where water soluble)
or dispersions and sterile powders for the extemporaneous
preparation of stei:ile injectable solutions or
dispersions. For »ntravenous administration., suitable
carriers include physiological saline, bacteriostatic
water, Cremophor EhTM (BASF; Parsippany, NJ) or phosphate
buffered saline (PBS). In. all cases, the composition
must be sterile and should. be fluid to the extent that
easy syringability exists. Tt must be stable under the
conditions of manui_acture and storage and must be
preserved against t:he contaminating action of
microorganisms such as bacteria and fungi. The carrier
can be a solvent oz- dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/I4201
the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial
and antifungal ageruts, for example, parabens,
chlorobutanol, pheruol, ascorbic acid, thimerosal, and the
like. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such
as mannitol, sorbit.ol, sodium chloride in the
composition. Prolc>nged absorption of the injectable
compositions can be: brought about by including in the
20 composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
Sterile inj~~ctable solutions can be prepared by
incorporating the active compound (e. g., a TlzO protein
or anti-T120 antibody) in the required amount in an
Z5 appropriate solvent: with one or a combination of
ingredients enumerated above, as required, followed by
filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a
sterile vehicle which contains a basic dispersion medium
20 and the required other ingredients from those enumerated
above. In the case: of sterile powders for the
preparation of stei:ile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active
25 ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
Oral compositions generally include an inert
diluent or an edible carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the
30 purpose of oral therapeutic administration, the active
compound can be incorporated with excipients and used in
the form of tablet:, troches, or capsules. Oral
compositions can a:Lso be prepared using a fluid carrier
for use as a mouthwash, wherein the compound in the fluid
35 carrier is applied orally and swished arid expectorated or
CA 02335585 2000-12-21
WO 99/67415 PCTlUS99/14201
-- 5$ -
swallowed. Pharmaceutically compatible binding agents,
and/or adjuvant mair,erials can be included as part of the
composition. The tablets, pills, capsules, troches and
the like can contain any of the following ingredients, or
compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as ;starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a
lubricant such as magnesium stearate or Sterotes; a
glidant such as co:Lloidal silicon dioxide; a sweetening
agent such as sucrose or saccharin; or a flavoring agent
such as peppermint, methyl. salicylate, or orange
flavoring. F'or administration by inhalation, the
compounds are delivered in the form of an aerosol spray
from a pressurized container or dispenser which contains
a suitable propell;~nt, e.g., a gas such as carbon
dioxide, or a nebu:lizer.
Systemic administration can also be by
transmucosal or transdermal means.. For transmucosal or
transdermal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art, and
include, for exatnp:le, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives.
Transmucosal administration can be accomplished through
the use of nasal sprays or suppositories. For
transdermal administration, the active compounds are
formulated into ointments, salves, gels, or creams as
generally known in the art.
The compounds can also be prepared in the form of
suppositories (e. g., with conventional suppository bases
such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
In one embodiment, the active compounds are
prepared with carriers that will protect the compound
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 59 -
against rapid elimination from.the body, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable,
biacompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, palyorthoesters, and polylactic acid. Methods
for preparation of such farmulations will be apparent to
those skilled in tine art. The materials can also be
obtained commercially from Alza Corporation and Nova
l0 Pharmaceuticals, Inc. Liposomal suspensians (including
liposomes targeted to infected cells with monoclonal
antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in
the art, for example, as described in U.S. Patent No.
4,522,811.
It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit
form as used herein refers to physically discrete units
suited as unitary dosages fox the subject to be treated;
each unit containing a predetermined quantity of active
campound 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.
The nucleic: acid molecules of the invention can be
inserted into vectors and used as gene therapy vectors.
Gene therapy vectors can be delivered to a subject by,
for example, intravenous :injection, local administration
(U. S. Patent 5,328,470) or by stereotactic injection
CA 02335585 2000-12-21
WO 99/67415 PCTIU899/14201
- 60 -
(see, e.g. , Chen ett al. (1.994) .Proc. Nail. Acad. Sci. USA
91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector
in an acceptable d:iluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded:
Alternatively, where the complete gene delivery vector
can be produced initact from recombinant cells, e.g.
retroviral vectors, the pharmaceutical preparation can
include one or more cells which produce the gene delivery
system.
The pharmaceutical compositions can be included in
a container, pack, or dispenser together with
instructions for administration.
V. Uses and Methods of the Invention
The nucleic' acid molecules, proteins, protein
homologues, and antibodies described herein can be used
in one or more of the fol7.owing methods: a) screening
assays; b) detecti~an assays (e. g., chromosomal mapping,
tissue typing, forensic biology), c) predictive medicine
(e. g., diagnostic .assays, prognostic assays, monitoring
clinical trials, a:nd pharmacogenomics): and d) methods of
treatment (e. g., therapeutic and prophylactic). T110
protein interacts with other cellular proteins and can
thus be used for (i) regulation of cellular proliferation
and (ii) regulation of cellular differentiation. The
isolated nucleic acid molecules of the invention can be
used to express T110 protein (e. g., via a recombinant
expression vector in a host cell in gene therapy
applications), to detect T110 mRNA (e. g., in a biological
sample) or a genetic lesion in a T110 gene, and to
modulate T110 activity. In addition, the T110 proteins
can be used to screen drugs or compounds which modulate
T110 activity or expression as well as to treat disorders
characterized by insufficient or excessive production of
CA 02335585 2000-12-21
WO 99!67415 PCT/US99/14201
- 61 -
T110 protein or production of T110 protein forms which
have decreased or aberrant activity compared to T110 wild
type protein. In <~ddition., the anti-T110 antibodies of
the invention can r>e used to detect and isolate T110
proteins and modulate T110 activity.
This invention further pertains to novel agents
identified by the above-described screening assays and
uses thereof for treatments as described herein.
A. Screenincx Assays
The invention provides a method (also referred to
herein as a "screening assay") for identifying
modulators, i.e., c:andidate or test compounds or agents
(e.g., peptides, peptidomimetics, small molecules or
other drugs) which bind to T110 proteins or have a
stimulatory or inh_Lbitory effect on, fox example, T110
expression or T110 activity.
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
T110 protein or po:Lypeptide or biologically active
portion thereof. '.Che test compounds of the present
invention can be obtained using any of the numerous
approaches in combinatorial library methods known in the
art, including: biological; libraries; spatially
addressable parallE~l 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 arf~ applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam
(1997} Anticancer Drug Des. 12:145) .
CA 02335585 2000-12-21
WO 99167415 PCT/US99/14201
- 62 -
_ Examples of: methods for. the synthesis of molecular
libraries can be f~aund in the art, for example in:
DeWitt et al. (199:3) Proc. Natl. Acad. Sci. U.S.A.
90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA
91:11422; Zuckerma:nn et a~.. (1994): J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et
al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et
al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and
Gallop et al. (1994) J. Med. Chem. 37:1233.
Libraries of compounds may be presented in
solution (e. g., Houghten (1992) Bio/Techniques 13:412-
421), or on beads (Lam (1991) Nature 354:82-84), chips
(Fodor (1993) Nature 364:555-556), bacteria (U. S. Patent
No. 5,223,409), spores (Patent Nos. 5,571,698; 5,403,484;
and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl.
Acad. Sci. LTSA 89:1865-1869) or phage (Scott and Smith
(1990) Science 249:386-390; Devlin (1990) Science
249:404-406; Cwirla et al,. (1990) Proc. Natl. Acad. Sci.
87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-
310) .
In one embodiment, an assay is a cell-based assay
in which a cell which expresses a membrane-bound form of
T110 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 T110
protein determined. The cell, for example, can be a
yeast cell or a cell of mammalian origin. Determining
the ability of the test compound to bind to the T110
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 T110 protein or
biologically active portion thereof can be determined by
detecting the labeled compaund in a complex. For
example, test compounds can be labeled with l2sl, 355, 1
ox 3H, either directly or indirectly, and the radioisotope
CA 02335585 2000-12-21
WO 99/67415 PCTIUS99/14201
- 63 -
detected by direct counting of .radioemmission or by
scintillation counting. Alternatively, test compounds
can be enzymatical7Ly 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 a preferred embodiment, the assay
comprises contacting a cell which expresses a membrane-
bound form of T110 protein., or a biologically active
portion thereof, on the cell surface with a known
compound which binds T110 to form an assay mixture,
contacting the assay mixture with a test compound, and
determining the ab:ility of the test compound to interact
with a T110 protein, wherein determining the ability of
the test compound i~o interact with a T110 protein
comprises determining the ability of the test compound to
preferentially bind to T110 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 T110 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., st:imulate or inhibit) the activity of the
T110 protein or biologically active portion thereof.
Determining the ability of the test compound to modulate
the activity of T1:10 or a biologically active portion
thereof can be accomplished, far example, by determining
the ability of the T110 protein to bind to or interact
with a T110 target molecu7.e.
As used herein, a ~~target molecule's is a molecule
with which a T110 ;protein binds or interacts in nature,
for example, a molecule on the surface of a cell which
expresses a T110 protein, a molecule on the surface of a
second cell, a molecule in the extracellular milieu, a
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 64 -
molecule associated with the internal surface of a cell
membrane or a cytoplasmic molecule. A T110 target
molecule can be a non-T110 molecule or a T110 protein or
polypeptide of the present invention. In one embodiment,
a T110 target molecule is a component of a signal
transduction pathway which facilitates transduction of an
extracellular signal (e. g., a signal generated by binding
of a compound to a membrane-bound T110 molecule or by
binding of a soluble form of T110 to a cellular receptor)
through the cell membrane and into the cell. The target,
for example, can be' a second intercellular protein which
has catalytic activity or a protein which facilitates the
association of downstream signaling molecules with T110.
Determining the ability of the T110 protein to
bind to or interact, with a T110 target molecule can be
accomplished by one of the methods described above fox
determining direct binding. In a preferred embodiment,
determining the ab_Llity of the T110 protein to bind to or
interact with a T110 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 detecaing induction of a cellular second
messenger of the target (a.g., intracellular Caz*,
diacylglycerol, IP3, etc.), detecting catalytic/enzymatic
activity of the target on an appropriate substrate,
detecting the induction of a reporter gene (e. g., a T110-
responsive regulatory element operably linked to a
nucleic acid encod_Lng a detectable marker, e.g.
luciferase), or detecting a cellular response, for
example, cellular <iifferen.tiation or cell proliferation.
In yet another embodiment, an assay of the present
invention is a cell-free assay comprising contacting a
T110 protein or biologically active portion thereof with
a test compound anti determining the ability of the test
compound to bind to the T110 protein or biologically
CA 02335585 2000-12-21
WO 99/67415 PCT/US99114201
- 65 -
active.portion thereof.(e.g., the extracellular domain of
T110). Binding of the test compound to the T110 protein
can be determined Either directly or indirectly as
described above. 7:n a preferred embodiment, the assay
includes contacting the T110 protein or biologically
active portion thex-eof with a known compound which binds
T110 to form an asp;ay mixture, contacting the assay
mixture with a test: compound, and determining the ability
of the test compound to interact with a T110 protein,
wherein determining the ability of the test compound to
interact with a T110 protein comprises determining the
ability of the test: compound to preferentially bind to
T110 or biological7.y active portion thereof as compared
to the known compound.
In another embodiment, an assay is a cell-free
assay comprising contacting T110 protein or biologically
active portion thereof with a test compound and
determining the ab~'~lity of the test compound to modulate
(e. g., stimulate or inhibit) the activity of the T110
protein or biologic: ally active portion thereof.
Determining the ab~~lity of the test compound to modulate
the activity of T1.~0 can be accomplished, for example, by
determining the ability of the T110 pratein to bind to a
T110 target molecu~e by one of the methods described
above for determin_Lng direct binding. In an alternative
embodiment, determining the ability of the test compound
to modulate the activity of TI10 can be accomplished by
determining the ab_Llity of the T110 protein to further
modulate a T110 target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as previously
described.
In yet another embodiment, the cell-free assay
comprises contacting the T110 protein or biologically
active portion thereof with a known compound which binds
CA 02335585 2000-12-21
WO 99167415 PCT/US99/14201
- 66 -
T110 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 T110 protein,
wherein determining the ability of the test compound to
interact with a T110 protein comprises determining the
ability of the T110 protein to preferentially bind to or
modulate the activ:ity of a. T110 target molecule .
The cell-free assays of the present invention are
amenable to use of both the soluble form or the membrane
bound form of T110.. In the case of cell-free assays
comprising the membrane-bound form of T110, it may be
desirable to utilize a solubilizing agent such that the
membrane-bound form of T110 is maintained in solution.
Examples of such solubiiizing agents include non-ionic
detergents such as n-octylglucoside, n-dodecylglucoside,
n-dodecylmaltoside,, octanoyl-N-methylglucamide, decanoyl-
N-methylglucamide, Triton' X-100, Triton~ X-11~, Thesit°,
Isotridecypoly(eth;rlene glycol ether)n, 3-[(3-cholamido-
propyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-
[(3-cholamidopropy:L)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO)" or N-dodecyl=N,N-dimethyl-3-ammonio-
1-propane sulfonate:.
In more than one embodiment of the above assay
methods of the pre:sent invention, it may be desirable to
immobilize either '.0110 or its target molecule to
facilitate separat:ion of complexed from uncomplexed forms
of one or both of the proteins, as well as to accommodate
automation of the assay. A test compound to T110, or
interaction of T110 with a. target molecule in the
presence and absenc=e of a candidate compound, can be
accomplished in an~~r vessel suitable for containing the
reactants. Examples of such vessels include microtitre
plates, test tubes;, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a
domain that allows one or both of the proteins to be
CA 02335585 2000-12-21
WO 99/67415 PCT/US99114201
- 67 -
bound to a matrix. For example, glutathione-S-
transferase/ T110 fusion proteins or glutathione-S-
transferase/target fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical; St. Louis,
MO) or glutathione derivatized microtitre plates, which
are then combined vuith the test compound or the test
compound and either the non-adsorbed target protein or
T110 protein, and t:he mixture incubated under conditions
conducive to complex formation (e. g., at physiological
conditions for salt: and pH). Following incubation, the
beads or microtitre: plate wells are washed to remove any
unbound components and complex formation is measured
either directly or indirectly, for example, as described
above. Alternatively, the complexes can be dissociated
from the matrix, and the level. of T110 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 T110 or its target
molecule can be immobilized utilizing conjugation of
biotin and streptavidin. Biotinylated T110 or target
molecules can be prepared from biotin-NHS (N-hydroxy-
succinimide) using techniques well known in the art
(e. g., biotinylation kit, Pierce Chemicals; Rockford,
IL), and immobilized in the wells of streptavidin-coated
96 well plates (Pierce Chemical). Alternatively,
antibodies reactive with T110 or target molecules but
which do not inter:Eere with binding of the T110 protein
to its target molenule can be derivatized to the wells of
the plate, and unbound target or T110 trapped in the
wells by antibody conjugation. 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 T110 or
target molecule, as well as enzyme-linked assays which
CA 02335585 2000-12-21
WfJ 99/67415 PCT/US99/14201
-- 68 -
rely on detecting an enzymatic activity associated with
the T110 or target molecule.
In another embodiment, modulators of T110
expression are identified in a method in which a cell is
contacted with a candidate compound and the expression of
T110 mRNA or protein in the cell is determined. The
level of expression. of T110 mRNA or protein in the
presence of the candidate compound is compared to the
level of expression. of T110 mRNA or protein in the
l0 absence of the candidate compound. The candidate
compound can then be identified as a modulator of T110
expression based an this comparison. Fox example, when
expression of T110 mRNA or protein is greater
(statistically significantly greater) in the presence of
the candidate compound than in its absence, the candidate
compound is identified as a stimulator of T110 mRNA or
protein expression. Alternatively, when expression of
T110 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 T110 m~RlVA or protein expression. The level
of T110 mRNA or protein expression in the cells can be
determined by methods described herein for detecting T110
mRNA or protein.
In yet another aspect of the invention, the T110
proteins can be used as "bait proteins" in a two-hybrid
assay or three hybrid assay (see, e.g., U.S. Patent No.
5,283,317; Zervos ea al. (1993) Cell 72:223-232; Madura
et al. (1993) J. Biol. Chew. 268:12046-12054; Bartel et
al. (1993) B.io/Tec.~hniques 14:920-924; Iwabuchi et al.
(1993) Oncogene 8:1.693-1696; and PCT Publication No. WO
94/10300), to identify other proteins, which bind to or
interact with T110 ("T110-:binding proteins" or "T110-by")
and modulate T110 activity. Such T110-binding proteins
are also likely to be involved in the propagation of
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
-~ 69 -
signals by the T110 proteins as, for example, upstream ar
downstream elements of the T110 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 T110 is fused to a
gene encoding the DL~A 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 vi.vo, forming an T110-dependent 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)
which 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 gene which encodes
the protein which interacts with T110.
This invention further pertains to novel agent s
identified by the above-described screening assays and
uses thereof for treatments as described herein.
B. Detection Assays
Portions or fragments of the cDNA sequences
identified herein (and the corresponding complete gene
sequences) can be used in numerous ways as polynucleotide
reagents. For example, these sequences can be used to:
(i) map their respective genes on a chromosome and, thus,
locate gene region; associated with genetic disease;
CA 02335585 2000-12-21
WO 99/674x5 PCT/US99/14201
- 70 -
(ii) identify an individual from a minute biological
sample (tissue typing} ; and (iii) aid in forensic
identification of a biological sample. These
applications are d<escribed in the subsections below.
1. Chromosome Mapping
Once the sequence (or a portion of the sequence}
of a gene has been isolated, this sequence can be used to
map the location of the gene on a chromosome.
Accordingly, T110 nucleic acid molecules described herein
or fragments thereof, can be used to map the location of
T110 genes on a chromosome. The mapping of the T110
sequences to chromosomes is an important .first step in
correlating these sequences with genes associated with
disease.
Briefly, T110 genes can be mapped to chromosomes
by preparing PCR p~_imers (preferably 15-25 by in length)
from the T110 sequE~nces. Computer analysis of T110
sequences can be used to rapidly select primers that do
not span more than one exon in the genomic DNA, thus
complicating the amplification process. These primers
can then be used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only
those hybrids cont<~ining the human gene corresponding to
the T110 sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing
somatic cells from different mammals (e.g., human and
mouse cells). As hybrids of human and mouse cells grow
and divide, they gradually lose human chromosomes in
random order, but ;retain the mouse chromosomes. By using
media in which mou:ae cells cannot grow (because they lack
a particular enzymes), but human cells can, the one human
chromosome that contains the gene encoding the needed
enzyme will be retained. By using various media, panels
of hybrid cell lincas can be established. Each cell line
CA 02335585 2000-12-21
WO 99/67415 PCTIUS99/14201
- 71 -
in a panel contains either a single human chromosome or a
small number of human chromosomes, and a full set of
mouse chromosomes, allowing easy mapping of individual
genes to specific human chromosomes. (D'hustachio et al.
(1983) Science 220:919-924). Somatic cell hybrids
containing only fr<~gments of human chromosomes can also
be produced by using human chromosomes with
translocations and deletions.
PCR mappings of somatic cell hybrids is a rapid
procedure for assigning a particular sequence to a
particular chromosome. Three or more sequences can be
assigned per day using a single thermal cycler. Using
the T110 sequences to design oligonucleotide primers,
sublocalization can be achieved with panels of fragments
from specific chromosomes. Other mapping strategies
which can similarly be used to map a T110 sequence to its
chromosome include in situ hybridization (described in
Fan et al. (1990) Pros. Nat3. Acad. Sci. USA 87:6223-27),
pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific
cDNA libraries.
Fluorescence in situ hybridization (FISH) of a DNA
sequence to a metaphase chromosomal spread can further be
used to provide a :precise chromosomal location in one
step. Chromosome spreads can be made using cells whose
division has been blocked in metaphase by a chemical,
e.g., colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then
stained with Giemsa. A pattern of light and dark bands
develops on each chromosome, so that the chromosomes can
be identified individually. The FISH technique can be
used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location
with sufficient signal intensity for simple detection.
CA 02335585 2000-12-21
WO 99167415 PCT/US991I4201
- 72 -
Preferably 1,000 bases, and more preferably 2,000 bases
will suffice to get good results at a reasonable amount
of time. For a review of this technique, see Verma et
al., (Human Chromosomes: A Manual of Basic Techniques
(Pergamon Press, New York, 1988)).
Reagents for chromosome mapping can be used
individually to mark a single chromosome or a single site
on that chromosome, or parcels of reagents can be used for
marking multiple sates and/or multiple chromosomes.
Reagents corresponding to noncoding regions of the genes
actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene
families, thus increasing the chance of cross
hybridizations during chromosomal mapping.
Once a seqmence has been mapped to a precise
chromosomal location, the physical position of the
sequence on the chromosome can be correlated with genetic
map data. (Such data axe found, for example, in V.
McKusick, Mendelian Inheritance in Man, available on-line
through Johns Hopk:ins University Welch Medical Library).
The relationship between genes and disease, mapped to the
same chromosomal region, can then be identified through
linkage analysis (co-inheritance of physically adjacent
genes), described :in, e.g., Egeland et al. (1987) Nature,
325:783-787.
Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease
associated with the T110 gene can be determined. If a
mutation is observed in same or all of the affected
individuals but not in any unaffected individuals, then
the mutation is li',~kely to be the causative agent of the
particular disease. Comparison of affected and
unaffected individuals generally involves first looking
for structural alterations in the chromasomes such as
deletions or trans:Iocations that are visible from
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 73 -
chromosome spreads or detectable using PCR based on that
DNA sequence. Ult=i.mately, complete sequencing of genes
from several indiv_Lduals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
2. Tissue Typing
The T110 sequences of the present invention can
also be used to ide~ntify individuals from minute
biological samples.. The United States military, for
example, is considering the use of restriction fragment
length polymorphism (RFLP) for identification of its
personnel. In thi;~ technique, an individual's genomic
DNA is digested with one or more restriction enzymes, and
probed on a Southern blot to yield unique bands for
identification. This method does not suffer from the
current limitations of ~~Dag Tags~a which can be lost,
switched, ar stolen; making positive identification
difficult. The sequences of the present invention are
useful as additional DNA markers far RFLP (described in
U.S. Patent 5,272,057).
Furthermore, the sequences of the present
invention can be u:~ed to provide an alternative technique
which determines the actual base-by-base DNA sequence of
selected portions c~f an individual's genome. Thus, the
T110 sequences described herein can be used to prepare
two PCR primers from the 5' and 3' ends of the sequences.
These primers can then be used to amplify an individual's
DNA and subsequently sequence it.
Panels of corresponding DNA sequences from
individuals, prepared in this manner, can provide unique
individual identifications, as each individual will have
a unique set of such DNA sequences due to allelic
differences. The ;sequences of the present invention can
be used to obtain ;such identification sequences from
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
_ 7
individuals and from tissue. The T110 sequences of the
invention uniquely represent portions of the human
genome. Allelic variation occurs to some degree in the
coding regions of these sequences, and to a greater
degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a
frequency of about once per each 500 bases. Each of the
sequences described herein can, to some degree, be used
as a standard against which DNA from an individual can be
compared for identification purposes. Because greater
numbers of polymorphisms occur in the noncoding regions,
fewer sequences arE: necessary to differentiate
individuals. The noncoding sequences of SEQ ID NO:1 can
comfortably provides positive individual identification
with a panel of perhaps 10 to 1,000 primers which each
yield a noncoding amplified sequence of 100 bases. If
predicted coding sE:quences, such as those in SEQ ID N0:3
are used, a more appropriate number of primers for
positive individual identification would be 500-2,000.
If a panel of reagents from T110 sequences
described herein is used to generate a unique
identification database for an individual, those same
reagents can later be used to identify tissue from that
individual. Using the unique identification database,
positive identification of the individual, living or
dead, can be made from extremely small tissue samples.
3. Use of Partial T110 Sequences in Forensic
Biolocty
DNA-based identification techniques can also be
used in forensic b~_ology. Forensic biology is a
scientific field ennploying genetic typing of bialagical
evidence found at a crime scene as a means far positively
identifying, for example, a perpetrator of a crime. To
make such an ident~:fication, PCR technology can be used
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
_ 75 _
to amplify DNA sequences taken from very small biological
samples such as ti:asues, e.g., hair or skin, or body
fluids, e.g., blood, saliva, or semen found at a crime
scene. The amplified sequence can then be compared to a
standard, thereby allowing identification of the origin
of the biological :sample.
The sequences of the present invention can be used
to provide polynuc7~_eotide reagents, e.g., PCR primers,
targeted to specif~Lc loci in the human genome, which can
enhance the reliability of DNA-based forensic
identifications by,, for example, providing another
'identification marker~~ (i.e. another DNA sequence that
is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns
formed by restriction enzyme generated fragments.
Sequences targeted to noncoding regions of SEQ ID N0:1
are particularly appropriate for this use as greater
numbers of polymorphisms occur in the noncoding regions,
making it easier to differentiate individuals using this
technique. Examples of polynucleotide reagents include
the T110 sequences or portions thereof, e.g., fragments
derived from the noncoding regions of SEQ ID NO:1 or SEQ
ID N0:5 having a length of at least 20 or 30 bases.
The T110 sequences described herein can further be
used to provide polynucleotide reagents, e.g., labeled or
labelable probes which can be used in, for example, an
in situ hybridization technique, to identify a specific
tissue, e.g., brai:n tissue. This can be very useful in
cases where a forensic pathologist is presented with a
tissue of unknown origin. Panels of such T110 probes can
be used to identify tissue by species and/or by organ
type.
In a similar fashion, these reagents, e.g., T110
primers or probes can be used to screen tissue culture
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 76 -
for contamination (i.e., screen for the presence of a
mixture of different types of cells in a culture).
C. Predictive Medicine
The present invention also pertains to the field
of predictive medicine in which diagnostic assays,
prognostic assays, pharmacogenomics, and monitoring
clinical trails are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates
to diagnostic assays for determining T110 protein and/or
nucleic acid expression as well as T110 activity, in the
context of a biological sample (e. g., blood, serum,
cells, tissue) to thereby determine whether an individual
is afflicted with ,~. disease or disorder, or is at risk of
developing a disorder, associated with aberrant T110
expression or activity. The invention also provides for
prognostic (or predictive) assays for determining whether
an individual is a?t risk of developing a disorder
associated with T1:10 protein, nucleic acid expression or
activity. Fox example, mutations in a T120 gene can be
assayed in a biological sample. Such assays can be used
for prognostic or ~~redictive purpose to thereby
prophylacticall.y great an individual prior to the onset
of a disorder characterized by or associated with T110
protein, nucleic a~~id expression or activity.
Another ash>ect of the invention provides methods
for determining T110 protein, nucleic acid expression or
T110 activity in a:n individual to thereby select
appropriate therapeutic or. prophylactic agents for that
individual (referred to herein as "pharmacogenomics").
Pharmacogenomics allows far the selection of agents
{e. g., drugs) for therapeutic or prophylactic treatment
of an individual based on the genotype of the individual
(e.g., the genotype of the individual examined to
CA 02335585 2000-12-21
WO 99/67415 PCTIUS99I14z01
determine the ability of the individual to respond to a
particular agent.)
Yet another aspect of the invention pertains to
monitoring the influence of agents (e. g., drugs or other
compounds) on the Esxpressi.on or activity of T110 in
clinical trials.
These and other agents are described in further
detail in the following sections.
1. Diagnostic Assays
An exemplary method for detecting the presence or
absence of T110 in a biological sample involves obtaining
a biological sample from a test subject and contacting
the biological sample with a compound or an agent capable
of detecting T110 ~arotein or nucleic acid (e. g., mRNA,
genomic DNA) that ~ancodes T110 protein such that the
presence of T110 is detected in the biological sample.- A
preferred agent fo:r detecting T110 mRNA or genomic DNA is
a labeled nucleic ;acid probe capable of hybridizing to
T110 mRNA or genom:ic DNA. The nucleic acid probe can be,
for example, a ful:L-length T110 nucleic acid, such as the
nucleic acid of SEy ID NO~ 1, 3, 5, or 7, or a portion
thereof, such as a;n oligonucleotide of at least 15, 30,
50, 100, 250 or 500 nucleotides in length and sufficient
to specifically hybridize under stringent conditions to
T110 mRNA or genomic DNA. Other suitable probes for use
in the diagnostic .assays of the invention are described
herein.
A preferred agent for detecting T110 protein is an
antibody capable of binding to T110 protein, preferably
an antibody with a detectable label. Antibodies can be
polyclonal, or more preferably, monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or Flab'}z} can
be used. The term "labeled", with regard to the probe or
antibody, is intended to encompass direct labeling of the
CA 02335585 2000-12-21
WO 99!67415 PCT/US99/14201
_ 78 _
probe or antibody by coupling (i.e., physically linking)
a detectable substance to the probe or antibody, as well
as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled.
Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary
antibody and end-labeling of a DNA probe with biotin such
that it can be detected with fluorescently labeled
streptavidin. The term "biological sample" is intended
to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids
present within a subject. That is, the detection method
of the invention can be used to detect T110 mRNA,
protein, or genomic DNA in a biological sample in vitro
as well as in vivo. For example, in vitro techniques for
detection of T110 mRNA include Northern hybridizations
and in situ hybridizations. In vitro techniques for
detection of T110 protein include enzyme linked
immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro
techniques for detection of T110 genomic DNA include
Southern hybridizations. Furthermore, ire vivo techniques
for detection of T110 protein include introducing into a
subject a labeled anti-T110 antibody. For example, the
antibody can be labeled with a radioactive marker whose
presence and location in a subject can be detected by
standard imaging techniques.
In one embodiment, the biological sample contains
protein molecules from the test subject. Alternatively,
the biological sample can contain mRNA molecules from the
test subject or genomic DNA molecules from the test
subject. A preferred biological sample is a peripheral
blood leukocyte sample isolated by conventional means
from a subject.
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 79 _
In another embodiment, the methods further involve
obtaining a contro:L biological sample from a control
subject, contacting the cantrol sample with a compound or
agent capable of dcatecting T110 protein, mRNA, or genomic
DNA, such that the presence of TI10 protein, mRNA or
genomic DNA is detected in the biological sample, and
comparing the presence of T110 protein, mF.NA or genomic
DNA in the control sample with the presence of T110
protein, mRNA or gE?nomic DNA in the test sample.
The invention also encompasses kits for detecting
the presence of T1:L0 in a biological sample (a test
sample). Such kit: can be used to determine if a subject
is suffering from or is at increased risk of developing a
disorder associated with aberrant expression of T110
(e.g., an immunological disorder). For example, the kit
can comprise a labeled compound or agent capable of
detecting T110 protein or mRNA in a biological sample and
means for determining the amount of T110 in the sample
(e. g., an anti-T110 antibody or an oligonucleotide probe
which binds to DNA encoding T110, e.g., SEQ ID N0:1, SEQ
ID N0:3, SEQ ID N0:5, or SEQ ID N0:7). Kits may also
include instruction for observing that the tested subject
is suffering from or is at risk of developing a disorder
associated with aberrant expression of T110 if the amount
of T110 protein or mRNA is above or below a normal level.
For antibocEy-based kits, the kit may comprise, for
example: (1) a first antibody (e. g., attached to a solid
support) which binds to T110 protein; and, optionally,
(2) a second, different antibody which binds to T110
protein or the firat antibody and is canjugated to a
detectable agent.
For oligonucleotide-based kits, the kit may
comprise, for example: (1) an oligonucleotide, e.g., a
detectably labelled oligonucleotide, which hybridizes to
CA 02335585 2000-12-21
WO 99!67415 PCT/US99l14201
- 80 -
a T110 nucleic aci<3 sequence or (2) a pair of primers
useful for amplifying a T110 nucleic acid molecule;
The kit may also comprise, e.g., a buffering
agent, a preservative, or a protein stabilizing agent.
The kit may also comprise components necessary for
detecting the detectable agent (e.g., an enzyme or a
substrate). The k:it may also contain a control sample or
a series of contro:L samples which can be assayed and
compared to the test sample contained. Each component of
the kit is usually enclosed within an individual
container and all of the various containers are within a
single package along with instructions for observing
whether the tested subject is suffering from or is at
risk of developing a disorder associated with aberrant
expression of T110.
2. Proc~nc>stic Assays
The method~~ described herein can furthermore be
utilized as diagnostic or prognostic assays to identify
subjects having or at risk of developing a disease or
disorder associated with aberrant T110 expression or
activity. For example, the assays described herein, such
as the preceding diagnostic assays or the following
assays, can be utilized to identify a subject having or
at risk of developing a disorder associated with T110
protein, nucleic acid expression or activity, e.g., a
cell proliferation disorder. Alternatively, the
prognostic assays can be utilized to identify a subject
having or at risk for developing such a disease or
disorder. Thus, the present invention provides a method
in which a test sample is obtained from a subject and
T110 protein or nucleic acid (e.g., mRNA, genomic DNA) is
detected, wherein the presence of T110 protein or nucleic
acid is diagnostic for a subject having or at risk of
developing a disease or disorder associated with aberrant
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 81 -
Tllo expression or activity. As used herein, a "test
sample" refers to a biological sample obtained from a
subject of interesi~. For example, a test sample can be a
biological fluid (Ea.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described
herein can be used to determine whether a subject can be
administered an agcsnt (e. g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate) to treat a disease or
disorder associated with aberrant T110 expression or
activity. For example, such methods can be used to
determine whether a subject can be effectively treated
with a specific agent or class of agents (e.g., agents of
a type which decrease T110 activity). Thins, the present
invention provides methods for determining whether a
subject can be effectively treated with an agent for a
disorder associated with aberrant T110 expression or
activity in which a test sample is obtained and T110
protein or nucleic acid is detected (e.g., wherein the
presence of T110 protein or nucleic acid is diagnostic
for a subject that can be administered the agent to treat
a disorder associated with aberrant T110 expression or
activity).
The method~> of the invention can also be used to
detect genetic lesions or mutations in a T110 gene,
thereby determining if a subject with the lesioned gene
is at risk for a disorder characterized by aberrant T110
expression or activity, e.g., aberrant cell proliferation
and/or differentiation. :Cn preferred embodiments, the
methods include detecting, in a sample of cells from the
subject, the presence or absence of a genetic lesion or
mutation characterized by at least one of an alteration
affecting the integrity of a gene encoding a T110-
protein, or the mis-expression of the T110 gene. For
example, such genetic lesions can be detected by
CA 02335585 2000-12-21
WO 99!67415 PCT/US99/14201
- 82 -
ascertaining the e:~istence of at least one of: 1) a
deletion of one or more nucleotides from a T110 gene; 2)
an addition of one or more nucleotides to a T110 gene; 3)
a substitution of one or more nucleotides of a T1I0 gene;
4) a chromosomal rE:arrangement of a T110 gene; 5) an
alteration in the :Level of a messenger RNA transcript of
a T110 gene; 6) aberrant modification of a T110 gene,
such as of the methylation pattern of the genomic DNA, 7)
the presence of a non-wild. type splicing pattern of a
messenger RNA transcript of a T110 gene, 8) a non-wild
type level of a T1:L0-protein, 9) allelic loss of a T110
gene, 10) inappropriate post-translations! modification
of a T110-protein, and 11) amplification of a T1I0 gene.
As described herein, there are a large number of assay
techniques known in the art which can be used for
detecting lesions :in a T110 gene.
In certain embodiments, detection of the lesion
involves the use o:E a probe/primer in a polymerase chain
reaction (PCR) (sen, e.g., U.S. Patent Nos. 4,683,195 and
4,683,202), such a;s anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR) (see,
e.g., Landegran et al. (1988) Scaence 241:1077-1080; and
Nakazawa et ai. (1994) Proc. NatZ. Acad. Sci. USA 91:360-
364), the latter o:E which can be particularly useful for
detecting point muitations in the T1I0-gene (see, e.g,,
Abravaya et al. (1995) Nucleic Acids Res. 23:675-682).
This method can include the steps of collecting a sample
of cells from a patient, isolating nucleic acid (e. g.,
genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or mare
primers which specifically hybridize to a T110 gene under
conditions such that hybridization and amplification of
the TI10-gene (if ;present) occurs, and detecting the
presence or absenc~' of an amplification product, or
detecting the size of the amplification product and
CA 02335585 2000-12-21
WO 99!67415 PCTIUS99/14201
- 83 -
comparing the length to a control sample. It is
anticipated that PCR and/or LCR may be desirable to use
as a preliminary amplification step in conjunction with
any of the techniques used for detecting mutations
described herein.
Alternative amplification methods include: self
sustained sequence replication (GuateZli et al. (1990)
Proc. Nail. Acad, Sci. USA 87:1874--1878), transcriptional
amplification system (Kwoh, et al. (1989) Proc. Natl.
Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi
et al. (1988} Bio/Technology 6:1197}, or any other
nucleic acid amplification method, followed by the
detection of the amplified molecules using techniques
well known to those of skill in the art. These detection
schemes are especially useful for the detection of
nucleic acid molecules if such molecules axe present in
very low numbers.
In an alternative embodiment, mutations in a T110
gene from a sample cell can be identified by alterations
in restriction enzyme cleavage patterns. Far example,
sample and control DNA is isolated, amplified
(optionally), digested with one or more restriction
endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in
fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use
of sequence specific ribozymes (see, e.g., U.B. Patent
No. 5,498,531) can be used to score for the presence of
specific mutations by development or loss of a ribozyme
cleavage site.
In other embodiments, genetic mutations in T110
can be identified by hybridizing a sample and control
nucleic acids, e.g., DNA or RNA, to high density arrays
containing hundreds or thousands of oligonucleotides
probes (Cronin et al. (1996) ~Iuman Mutation 7:244-255;
CA 02335585 2000-12-21
WO 99167415 PCT/US99I14201
- 84 -
Kozal et al. (1996;) Nature Medicine 2:753-759). For
example, genetic mutations in T110 can be identified in
two-dimensional arrays containing light-generated DNA
probes as described in Cronin et al. supra. Briefly, a
first hybridization array of probes can be used to scan
through long stretches of DNA in a sample and control to
identify base than<~es between the sequences by making
linear arrays of sequential overlapping probes. This
step allows the identification of point mutations. This
step is followed by a secand hybridization array that
allows the characterization of specific mutations by
using smaller, specialized probe arrays complementary to
all variants or mutations detected. Each mutation array
is composed of parallel probe sets, one complementary to
the wild-type gene and the other complementary to the
mutant gene.
In yet another embodiment, any of a variety of
sequencing reactions known in the art can be used to
directly sequence 'the T110 gene and detect mutations by
comparing the sequence of the sample T110 with the
corresponding wild-type (control) sequence. Examples of
sequencing reactions include those based on techniques
developed by Maxim and Gilbert ((1977) Pr~c. Natl. Acad.
Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad.
Sci. USA 74:5463). It is- also contemplated that any of a
variety of automated sequencing procedures can be
utilized when performing the diagnostic assays ((1995)
Bio/Techniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT Publication No. WO 94/16101;
Cohen et al. (1996) Adv. C'hromatogr. 36:127-162; and
Griffin et al. {1993) Appl. Biochem. Biotechnol. 38:147-
159) .
Other methods for detecting mutations in the T110
gene include methods in which protection from cleavage
agents is used to ;detect mismatched bases in RNA/RNA or
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 85 -
RNA/DNA heteroduple:xes . (Myers e.t al. (1985) Science
230:1242). In genE:ral, the technique of "mismatch
cleavage" entails providing heteroduplexes formed by
hybridizing (labeled) RNA or DNA containing the wild-type
T110 sequence with potentially mutant RNA or DNA obtained
from a tissue sample. The double-stranded duplexes are
treated with an agent which cleaves single-stranded
regions of the dup7.ex such as which will exist due to
basepair mismatches between the control and sample
strands. RNA/DNA duplexes can be treated with RNase to
digest mismatched regions, and DNA/DNA hybrids can be
treated with S1 nuclease to digest mismatched regions.
In other embodiment: s, either DNA/DNA ar RNA/DNA duplexes
can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions.
After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing
polyacrylamide gel~~ to determine the site of mutation.
See, e.g., Cotton eat al (1988) Proc. Nail Acad Sci USA
85:4397; Saleeba et: al (1992) Methods Enzymol. 217:286-
295. In a preferre=d embodiment, the control DNA or RNA
can be labeled for detection.
In still another embodiment, the mismatch cleavage
reaction employs one or more proteins that recognize
mismatched base pairs in double-stranded DNA (so called
"DNA mismatch repair" enzymes) in defined systems for
detecting and mapping point mutations in T110 cDNAs
obtained from samp:Les of cells. For example, the mutt
enzyme of E. coli cleaves A at G/A mismatches and the
thymidine DNA glycosylase from HeLa cells cleaves T at
G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-
1662). According i~o an exemplary embodiment, a probe
based on a T110 sequence, e.g., a wild-type T110
sequence, is hybridized to a cDNA or other DNA product
from a test cells;l. The duplex is treated with a DNA
CA 02335585 2000-12-21
WO 99/67415 PCTIU899/142D1
- 86 -
mismatch repair en;ayme,. and the cleavage products, if
any, can be detected from electrophoresis protocols or
the like. Gee, e.<3., U.S. Patent No. 5,459,039.
In other embodiments, alterations in
electrapharetic mobility will be used to identify
mutations in T110 <~enes. For example, single strand
conformation polymorphism (SSCP} may be used to detect
differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989} Proc.
Natl. Acad. Sci US.~ 86:276; see also Cotton (1993)
Mutat. Res. 285:12!x-144; I~ayashi (1992) Genet. Anal.
Tech. Appl. 9:73-7!a). Single-stranded DNA fragments of
sample and control T110 nucleic acids will be denatured
and allowed to renature. The secondary structure of
single--stranded nucleic acids varies according to
sequence, and the :resulting alteration in electrophoretic
mobility enables the detection of even a single base
change. The DNA fragments may be labeled or detected
with labeled probe;. The sensitivity of the assay may be
enhanced by using RNA (rather than DNA), in which the
secondary structure is more sensitive to a change in
sequence. In a preferred embodiment, the subject method
utilizes heterodup:lex analysis to separate double
stranded heterodupaex molecules an the basis of changes
in electrophoretic mobility (Keen et al. (1991) Trends
Genet 7:5).
In yet another embodiment, the movement of mutant
or wild-type fragments in polyacrylamide gels containing
a gradient of denaturant is assayed using denaturing
gradient gel electrophoresis (DGGE) (Myers et al. (1985}
Nature 313:495). lahen DGGE is used as the method of
analysis, DNA will be modified to insure that it does not
completely denature, for example by adding a GC clamp of
approximately 40 by of high-melting GC-rich DNA by PCR.
In a further embodiment, a temperature gradient is used
CA 02335585 2000-12-21
WO 99/b7415 PCTIUS99/14201
_ 87 _
in place of a denat:uring gradient to identify differences
in the mobility of control. and sample DNA (Rosenbaum and
Reissner (1987) Bic~phys Chem 265:12753) .
Examples of other techniques for detecting point
mutations include, but are not limited to, selective
oligonucleotide hybridization, selective amplification,
or selective prime:r extension. For example,
oligonucleotide primers may be prepared in which the
known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization
only if a perfect match is found (Saiki et al. (1986)
Nature 324:163}; Saiki et al. (1989) Proc. Natl Acad. Sci
USA 86:6230). Such. allele specific oligonucleotides are
hybridized to PCR amplified target DNA or a number of
different mutations when the oligonucleotides are
attached to the hybridizing membrane and hybridized with
labeled target DNA.
Alternatively, allele specific amplification
technology which depends on selective PCR amplification
may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific
amplification may carry the mutation of interest in the
center of the malecule (so that amplification depends on
differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res: 17:2437-2448) or at the extreme 3' end of one
primer where, under appropriate conditions, mismatch can
prevent or reduce polymerise extension (Prossner (1993)
Tibtech 11:238). In addition, it may be desirable to
introduce a novel restriction site in the region of the
mutation to create cleavage-based detection (Gasparini et
al. {1992) MoI. Cell Probes 6:1). It is anticipated that
in certain embodiments amplification may also be
performed using Taq ligase for amplification (Barany
{1991) Proc. Natl. Acid. Sci USA 88:189). In such cases,
ligation will occur only if there is a perfect match at
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
_ $$ _
the 3'_end of the !~° sequence making it possible to
detect the presenc<~ of a known mutation at a specific
site by looking fo=r the presence or absence of
amplification.
The methods. described herein may be performed, for
example, by utilizing pre-packaged diagnostic kits
comprising at leasi~ one probe nucleic acid or antibody
reagent described herein, which may be conveniently used,
e.g., in clinical wettings. to diagnose patients
exhibiting symptom; or family history of a disease or
illness involving a T110 gene.
Furthermore:, any cell type or tissue, preferably
peripheral blood leukocytes, in which T110 is expressed
may be utilized in the prognostic assays described
herein.
3. Pharma.cogenomics
Agents, or modulators which have a stimulatory or
inhibitory effect on T110 activity (e. g., T110 gene
expression) as identified by a screening assay described
herein can be administered to individuals to treat
(prophylactically or therapeutically) disorders (e.g., a
proliferative disorder} associated with aberrant T110
activity. In conjunction with such treatment, the
pharmacogenomics (:i.e., the study of the relationship
between an individual's genotype and that individual's
response to a foreign compound or drug) of the individual
may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic
failure by altering the relation between dose and blood
concentration of t:he pharmacologically active drug. Thus,
the pharmacogenomics of the individual permits the
selection of effective agents (e.g., drugs) for
prophylactic or therapeutic treatments based on a
consideration of t:he individual's genotype. Such
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 89 -
pharmacogenomics c,an further be used to determine
appropriate dosages and therapeutic regimens.
Accordingly, the activity of T110 protein, expression of
T120 nucleic acid, or mutation content of T110 genes in
an individual can be determined to thereby select
appropriate agents) for therapeutic or prophylactic
treatment of the individual.
Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to
altered drug disposition and abnormal action in affected
persons. See, e.g., Linder (1997) Clin. Chern. 43(2):254-
266. In general, two types of pharmacogenetic conditions
can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body
Z5 are referred to as "altered drug action" conditions.
Genetic conditions transmitted as single factors altering
the way the body acts on drugs axe referred to as
"altered drug metabolism" conditions. These
pharmacogenetic conditions can occur either as rare
defects or as polymorphisms. For example, glucose-6-
phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical
complication is haemolysis after ingestion of oxidant
drugs {anti-malarials, sulfonamides, analgesics,
nitrofurans) and consumption of fava beans.
As an illu~~trative embodiment, the activity of
drug metabolizing enzymes is a major determinant of both
the intensity and duration of drug action. The discovery
of genetic polymorphisms of drug metabolizing enzymes
(e. g., N-acetyltransferase 2 (NAT 2) and cytochrome P450
enzymes CYP2D6 and CYP2C19) has provided an explanation
as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious
toxicity after taking the standard and safe dose of a
drug. These polym.orphisms are expressed in two
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
- 90 -
phenotypes in the ;population, the extensive metabolizer
(EM) and poor meta'.bolizer (PM) . The prevalence of PM is
different among different populations. For example, the
gene coding far CY:P2D6 is highly polymorphic and several
mutations have been identified in PM, which all lead to
the absence of functional CYP2D6. Poor metabolizers of
CYP2D6 and CYP2C19 quite frequently experience
exaggerated drug response and side effects when they
receive standard doses. If a metabolite is the active
therapeutic moiety, PM Shaw no therapeutic response, as
demonstrated for t:he analgesic effect of codeine mediated
by its CYP2D6-formed metabolite morphine. The other
extreme are the so called ultra-rapid metabolizers who do
not respond to standard doses. Recently, the molecular
basis of ultra-rapid metabolism has been identified to be
due to CYP2D6 gene amplification.
Thus, the ~~ctivity of T110 protein, expression of
T110 nucleic acid, or mutation content of T110 genes in
an individual can :be determined to thereby select
appropriate agents) far therapeutic or prophylactic
treatment of the individual. In addition,
pharmacogenetic studies can be used to apply genotyping
of polymorphic alleles encoding drug-metabolizing enzymes
to the identification of an individual's drug
responsiveness phenotype. This knowledge; when applied
to dosing or drug selection, can avoid adverse reactions
or therapeutic failure and thus enhance therapeutic or
prophylactic efficiency when treating a subject with a
T110 modulator, such as a modulator identified by one of
the exemplary screening assays described herein.
4. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e. g., drugs,
compounds) on the expression or activity of T110 (e. g.,
the ability to modulate aberrant cell proliferation
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
_ 91 _
and/or. differentiat:ion), ca.n be -applied not only in basic
drug screening, but: also in clinical trials. For
example, the effectiveness of an agent, as determined by
a screening assay as described herein to increase T110
gene expression, increase protein levels, or upregulate
T110 activity, can be moni.tared in clinical trials of
subjects exhibiting decreased T110 gene expression,
decreased protein :Levels, or downregulated T110 activity.
Alternatively, the effectiveness of an agent, as
determined by a sc.~reening assay, to decrease T110 gene
expression, decreaae protein levels, or downregulate T110
activity, can be monitored in clinical trials of subjects
exhibiting increased T110 gene expression, increased
protein levels, or upregulated T110 activity. In such
clinical trials, the expression or activity of T110 and,
preferably, other genes that have been implicated in, for
example, a cellular proliferation disorder can be used as
a marker.
For example:, and not by way of limitation, genes,
including T110, that are modulated in cells by treatment
with an agent (e. g., compaund, drug or small molecule)
which modulates T110 activity (e.g., as identified in a
screening assay described herein) can be identified.
Thus, to study the effect of agents on cellular
-proliferation disorders, for examples in a clinical
trial, cells can be isolated and RNA prepared and
analyzed for the levels of expression of T110 and other
genes implicated in the disorder. The levels of gene
expression (i.e., a gene expression pattern) can be
quantified by Northern blot analysis or RT-PCR, as
described herein, ar alternatively by measuring the
amount of protein produced, by one of the methods as
described herein, or by measuring the levels of activity
of T110 or other genes. :Ln this way, the gene expression
pattern can serve as a marker, indicative of the
CA 02335585 2000-12-21
WO 99/67415 PCTIUS99/14201
- 92 -
physiological response. of the cells to the agent.
Accordingly, this :response state may be determined
before, and at various paints during, treatment of the
individual with the agent
In a preferred embodiment, the present invention
provides a method for monitoring the effectiveness of
treatment of a subject with an agent (e. g., an agonist,
antagonist, peptid~omimetic:, protein, peptide, nucleic
acid, small molecule, or other drug candidate identified
by the screening assays described herein) comprising the
steps of ti) obtaining a pre-administration sample from a
subject prior to administration of the agent; (ii)
detecting the level of expression of a T110 protein,
mRNA, or genomic D:I~A in the preadministration sample;
(iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression
or activity of the T110 protein, mRNA, ar genomic DNA in
the post-administration samples; (v) comparing the level
of expression or activity of the T110 protein, mRNA, or
genomic DNA in the pre-administration sample with the
T110 protein, m'RNA, or genomic DNA in the post
administration sample or samples; and (vi) altering the
administration of the agent to the subject accordingly.
For example, increased administration of the agent may be
desirable to increase the expression or activity of T110
to higher levels than detected, i.e., to increase the
effectiveness of the ageni~. Alternatively, decreased
administration of the agent may be desirable to decrease
expression or activity of T110 to lower levels than
detected, i.e., to decrease the effectiveness of the
agent.
C. Methods of Treatment
The presents invention provides for bath
prophylactic and therapeutic methods of treating a
CA 02335585 2000-12-21
WO 99/67415 PCT/US99I14201
- 93 -
subject at risk of (or.suscepti.ble to) a disorder or
having a disorder associated with aberrant T110
expression or activity. Such disorders include
neoplasia, inappropriate angiogenesis, or inappropriate
tissue regeneration.
Z. Prophylactic Methods
In one aspect, the invention provides a method for
preventing in a subject, a disease or condition
associated with an aberrant T110 expression or activity,
by administering to the subject an agent which modulates
T110 expression or at least one T110 activity. Subjects
at risk for a disease which is caused or contributed to
by aberrant T110 e:~pression or activity can be identified
by, for example, any or a combination of diagnostic or
prognostic assays as described herein. Administration of
a prophylactic agent can occur prior to the manifestation
of symptoms characi:eristic of the T110 aberrancy, such
that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on
the type of T110 aberrancy, for example, a T110 agonist
or T110 antagonist agent can be used for treating the
subject. The appropriate agent can be determined based
on screening assays described herein.
2. Therax~eutic Methods
Another aspect of the invention pertains to
methods of modulating T110 expression or activity for
therapeutic purposes. The modulatory method of the
invention involves contacting a cell with an agent that
modulates one or more of the activities of T110 protein
activity associated with the cell. An agent that
modulates T110 protein activity can be an. agent as
described herein, such as a nucleic acid or a protein, a
naturally-occurring cognate ligand of a T110 protein, a
CA 02335585 2000-12-21
WO 99/67415 PCTIUS99/I420I
- 94 -
peptide, a T110 peptidomimetic,. or other small molecule.
In one embodiment, the agent stimulates one or more of
the biological activities of T110 protein. Examples of
such stimulatory agents include active T110 protein and a
nucleic acid molecule encoding T110 that has been
introduced into thf: cell. In another embodiment, the
agent inhibits one or more of the biological activities
of T110 protein. Examples of such inhibitory agents
include antisense '.0110 nucleic acid molecules and anti-
T110 antibodies. '.Chese madulatory methods can be
performed in vitro (e.g., by culturing the cell with the
agent) or, alternai~iveiy, in vi v~ (e.g, by administering
the agent to a sub_ject). As such, the present invention
provides methods o~ treating an individual afflicted with
a disease or disorder characterized by aberrant
expression or activity of a T110 protein or nucleic acid
molecule. In one embodiment, the method involves
administering an agent (e.g., an agent identified by a
screening assay de:~cribed herein), or combination of
agents that modulai~es {e.g., upregulates or
downregulates) T110 expression or activity. In another
embodiment, the mei~hod involves administering a T110
protein or nucleic acid molecule as therapy to compensate
for reduced or aberrant T110 expression or activity.
Stimulation of T110 activity is desirable in
situations in which T110 is abnormally downregulated
and/or in which increased T110 activity is likely to have
a beneficial effect. Conversely, inhibition of T110
activity is desira3ale in situations in which T110 is
abnormally upregul;~ted and/or in which decreased T110
activity is likely to have a beneficial effect.
This invention is further illustrated by the
following examples which should not be construed as
limiting. The contents of all references, patents and
CA 02335585 2000-12-21
WO 99167415 PCT/US99114201
- 95 -
published patent applications cited throughout this
application are hereby incorporated by reference.
EXAMPLES
Example 1: Isolation and Characterization of Human T110
cDNAs
A cDNA library was prepared from polyA mRNA
isolated from ratPCl2 cel7_s (PC6-3 subline) that had been
cultured in the absence of neurotrophic factors (NGF) for
12 hours. Random 5' sequencing yielded a single clone
with homology to tike D. mPlanogaster fj gene. This
partial rat clone was used to screen mouse and human
fetal brain cDNA libraries. These screens have yielded
clones containing mouse T110 and human T110.
Complete sequencing of the human T110 clone
revealed an approximately 2.4 kb cDNA insert with a 1311
base pair open reading frame predicted to encode a novel
secreted protein, i.e., human T110. Complete sequencing
of the mouse T110 ~~lone revealed an approximately 2.1 kb
cDNA insert with a 1350 base pair open reading frame
predicted to encode a novel secreted protein, i.e., mouse
T110. The mouse a:nd human protein sequences are about
85o identical. The major region of divergence is towards
the N-terminus.
Figure 6 depicts the cDNA sequence {SEQ ID NO:1)
and predicted amino acid sequence (SEQ TD N0:4) of a
potential alternative human T110 translation product.
The open reading frame extends from nucleotide 2 to 1411
of SEQ ID N0:1) .
Figure 8 depicts the cDNA sequence (SEQ ID N0:5)
and predicted amino acid sequence (SEQ ID N0:8) of a
potential alternative murine T110 translation product.
The open reading frame extends from nucleotide 1 to 1452
of SEQ ID N0:5.
CA 02335585 2000-12-21
WO 99167415 PCT/US991142~1
- 96 -
Example 2: Distribution of T110 mRNA in Human Tissues
The expres.~ion of T110 was analyzed using Northern
blot hybridization. In rat, the Northern blot analysis
of adult tissues showed highest expression in brain and
kidney. Expression was also observed in heart and lung.
No mRNA was detected in spleen, liver, skeletal muscle or
testis.
To examine the tissue distribution of human T110,
the rat partial cDa~A sequence was used as a probe for the
Northern blot analysis. The cDNA was radioactively
labeled with 32P-dC'TP using the Prime-It kit (Stratagene;
La Jolla, CA) according to the instructions of the
supplier. Filters containing human mRNA (MTNI and MTNII:
Clontech; Palo Alto, CA) were probed in ExpressHyb
hybridization solution (Clontech, Palo Alta, CA) and
washed at high stringency according to manufacturer°s
recommendations.
These stud~~:es revealed that human T110 was
expressed as an approximately 2.4 kilobase transcript at
highest level in brain, heart, placenta, and pancreas.
Lower levels of transcript were seen in liver, skeletal
muscle, and kidney. Transcript was not detected in lung.
Embryonic expression was seen in week 8-9 fetus and week
20 liver and spleen mixed tissue.
In situ expression assays on mouse embryos
revealed that T110 is expressed in the nervous system.
In adult mice, in situ expression assays revealed that
T110 is expressed in discrete regions of the brain,
including the cerebellum and olfactory bulb, and in the
non-islet cells of the pancreas.
Example 3: Characterization of T110 Proteins
The human '.0110 cDNA (Figure 1; SEQ ID N0:1)
isolated as described above encodes a 437 amino acid
protein (Figure 1; SEQ ID N0:2). A hydrapathy plot of
CA 02335585 2000-12-21
WO 99167415 PCTIUS99114201
T110 is presented in Figure 2.. This plot shows the
presence of a signal sequence (amino acids 1-28) and a
hydrophobic region. that may indicate a transmembrane
domain (amino acid. 7-30) that acts as an internal signal
sequence.
Figure 7 i;~ a plot showing predicted structural
features of a potential alternative human T110 protein
(SEQ ID N0:4). This figure shows predicted alpha helix
regions (Garnier-R:obson and Chou-Fasman), predicted beta
sheet regions (Gamier-Robson and Chou-Fasman), predicted
turn regions (Gamier-Robson and Chou-Fasman), predicted
coil regions (Gamier-Robson), predicted hydrophilicity,
predicted alpha amphipathic regions (Eisenberg) predicted
beta amphipathic regions (Eisenberg), predicted flexible
regions (Karplus-Schultz), predicted antigenic index
(Jameson-Wolf), and surface probability (Emini).
A sequence alignment of human T110 protein and
D. melanagaster fj protein, as shown in Figure 6, reveals
that both proteina are of similar size, contain a single
predicted hydrophobic region as the transmembrane and
internal signal sequence, and include a large
extracellular dorr~iin with two pairs of conserved cysteine
residues. In this; alignment, which includes gaps, the
proteins are 20.7z> identical and 35.9°s similar.
Mature human T110 has a predicted MW of 48 kDa,
not including post:-translational modifications.
A secretion assay revealed that T110 is a secreted
protein. It may be secreted using a signal peptide
(amino acids 1-28) or a transmembrane region (amino acids
7-30) that acts a~~ an internal signal sequence.
Example 4: Pre oration of T110 Proteins
Recombinant T110 can be produced in a variety of
expression systems. For example, the mature T110 peptide
can be expressed as a recombinant glutathione-S-
CA 02335585 2000-12-21
WO 99/67415 PCT/US99/14201
_ g8 _
transferase (GST) j'usion protein in E. coli and the
fusion protein can be isolated and characterized.
Specifically, as described. above, Tllo can be fused to
GST and this fusion protein can be expressed in E. coli
strain PEB199. Expression of the GST-T110 fusion protein
in PEB199 can be induced with IPTG. The recombinant
fusion protein can be purified from crude bacterial
Iysates of the induced PEB199 strain by affinity
chromatography on c~lutathione beads.
Equivalents
Those skilled in the art will recognize, or be
able to ascertain using no more than routine
experimentation, many equivalents to the specific
embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the
following claims.
What is claimed is.:
