THERAPEUTIC POLYPEPTIDES, NUCLEIC ACIDS ENCODING SAME, AND METHODS OF USE

POLYPEPTIDES THERAPEUTIQUES, ACIDES NUCLEIQUES CODANT POUR CES POLYPEPTIDES ET METHODES D'UTILISATION ASSOCIEES

English Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides.
Also disclosed are polypeptides encoded by these nucleic acid sequences, and
antibodies that immunospecifically bind the polypeptide, as well as
derivatives, variants, mutants, or fragments of the novel polypeptide,
polynucleotide, or antibody specific to the polypeptide. Vectors, host cells,
antibodies and recombinant methods for producing the polypeptides and
polynucleotides, as well as methods for using same are also included. The
invention further discloses therapeutic, diagnostic and research methods for
diagnosis, treatment, and prevention of disorders involving any one of these
novel human nucleic acids and proteins.

French Abstract

L'invention concerne des séquences d'acides nucléiques codant pour de nouveaux polypeptides. L'invention concerne également des polypeptides codés par ces séquences d'acides nucléiques, ainsi que des anticorps qui se lient de façon immunospécifique à ces polypeptides, de même que des dérivés, variants, mutants ou fragments de ces polypeptides, ces polynucléotides ou ces anticorps spécifiques à ces polypeptides. L'invention concerne par ailleurs des vecteurs, des cellules hôtes, des anticorps et des procédés recombinants pour produire ces polypeptides et polynucléotides, ainsi que des méthodes d'utilisation de ceux-ci. L'invention concerne enfin des méthodes thérapeutiques et diagnostiques et des méthodes de recherche destinées au diagnostic, au traitement et à la prévention de troubles liés à ces protéines et ces acides nucléiques humains.

Claims

CLAIMS

What is claimed is:

1. An isolated polypeptide comprising the mature form of an amino acid
sequenced
selected from the group consisting of SEQ ID NO:2n, wherein n is an integer
between 1
and 606.
2. An isolated polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 606.
3. An isolated polypeptide comprising an amino acid sequence which is at least
95%
identical to an amino acid sequence selected from the group consisting of SEQ
ID NO:2n,
wherein n is an integer between 1 and 606.
4. An isolated polypeptide, wherein the polypeptide comprises an amino acid
sequence comprising one or more conservative substitutions in the amino acid
sequence
selected from the group consisting of SEQ ID NO:2n, wherein n is an integer
between 1
and 606.
5. The polypeptide of claim 1 wherein said polypeptide is naturally occurring
6. A composition comprising the polypeptide of claim 1 and a carrier.
7. A kit comprising, in one or more containers, the composition of claim 6.
8. The use of a therapeutic in the manufacture of a medicament for treating a
syndrome associated with a human disease, the disease selected from a
pathology
associated with the polypeptide of claim 1, wherein the therapeutic comprises
the
polypeptide of claim 1.
9. A method for determining the presence or amount of the polypeptide of
claim 1 in a sample, the method comprising:
(a) providing said sample;
(b) introducing said sample to an antibody that binds immunospecifically to
the
polypeptide; and
(c) determining the presence or amount of antibody bound to said polypeptide,
thereby determining the presence or amount of polypeptide in said sample.
10. A method for determining the presence of or predisposition to a disease
associated
with altered levels of expression of the polypeptide of claim 1 in a first
mammalian subject,
the method comprising:
a) measuring the level of expression of the polypeptide in a sample from the
first mammalian subject; and
b) comparing the expression of said polypeptide in the sample of step (a) to
the
expression of the polypeptide present in a control sample from a second
mammalian subject known not to have, or not to be predisposed to, said
disease,

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wherein an alteration in the level of expression of the polypeptide in the
first subject as
compared to the control sample indicates the presence of or predisposition to
said disease.
11. A method of identifying an agent that binds to the polypeptide of claim 1,
the
method comprising:
(a) introducing said polypeptide to said agent; and
(b) determining whether said agent binds to said polypeptide.
12. The method of claim 11 wherein the agent is a cellular receptor or a
downstream
effector.
13. A method for identifying a potential therapeutic agent for use in
treatment of a
pathology, wherein the pathology is related to aberrant expression or aberrant
physiological
interactions of the polypeptide of claim 1, the method comprising:
(a) providing a cell expressing the polypeptide of claim 1 and having a
property
or function ascribable to the polypeptide;
(b) contacting the cell with a composition comprising a candidate substance;
and
(c) determining whether the substance alters the property or function
ascribable
to the polypeptide;
whereby, if an alteration observed in the presence of the substance is not
observed when the
cell is contacted with a composition in the absence of the substance, the
substance is
identified as a potential therapeutic agent.
14. A method for screening for a modulator of activity of or of latency or
predisposition
to a pathology associated with the polypeptide of claim 1, said method
comprising:
(a) administering a test compound to a test animal at increased risk for a
pathology associated with the polypeptide of claim 1, wherein said test
animal recombinantly expresses the polypeptide of claim 1;
(b) measuring the activity of said polypeptide in said test animal after
administering the compound of step (a); and
(c) comparing the activity of said polypeptide in said test animal with the
activity of said polypeptide in a control animal not administered said
polypeptide, wherein a change in the activity of said polypeptide in said test
animal relative to said control animal indicates the test compound is a
modulator activity of or latency or predisposition to, a pathology associated
with the polypeptide of claim 1.
15. The method of claim 14, wherein said test animal is a recombinant test
animal that
expresses a test protein transgene or expresses said transgene under the
control of a
promoter at an increased level relative to a wild-type test animal, and
wherein said
promoter is not the native gene promoter of said transgene.
16. A method for modulating the activity of the polypeptide of claim 1, the
method
comprising contacting a cell sample expressing the polypeptide of claim 1 with
a
compound that binds to said polypeptide in an amount sufficient to modulate
the activity of
the polypeptide.
17. A method of treating or preventing a pathology associated with the
polypeptide of
claim 1, the method comprising administering the polypeptide of claim 1 to a
subject in

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which such treatment or prevention is desired in an amount sufficient to treat
or prevent the
pathology in the subject.
18. The method of claim 17, wherein the subject is a human.
19. A method of treating a pathological state in a mammal, the method
comprising
administering to the mammal a polypeptide in an amount that is sufficient to
alleviate the
pathological state, wherein the polypeptide is a polypeptide having an amino
acid sequence
at least 95% identical to a polypeptide comprising the amino acid sequence
selected from
the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and
606 or a
biologically active fragment thereof.
20. An isolated nucleic acid molecule comprising a nucleic acid sequence
selected from
the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and
606.
21. The nucleic acid molecule of claim 20, wherein the nucleic acid molecule
is
naturally occurring.
22. A nucleic acid molecule, wherein the nucleic acid molecule deters by a
single
nucleotide from a nucleic acid sequence selected from the group consisting of
SEQ ID NO:
2n-1, wherein n is an integer between 1 and 606.
23. An isolated nucleic acid molecule encoding the mature form of a
polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:2n,
wherein n is an integer between 1 and 606.
24. An isolated nucleic acid molecule comprising a nucleic acid selected from
the group
consisting of 2n-1, wherein n is an integer between 1 and 606.
25. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule
hybridizes under stringent conditions to the nucleotide sequence selected from
the group
consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 606, or a
complement of said nucleotide sequence.
26. A vector comprising the nucleic acid molecule of claim 20.
27. The vector of claim 26, further comprising a promoter operably linked to
said
nucleic acid molecule.
28. A cell comprising the vector of claim 26.
29. An antibody that immunospecifically binds to the polypeptide of claim 1.
30. The antibody of claim 29, wherein the antibody is a monoclonal antibody.
31. The antibody of claim 29, wherein the antibody is a humanized antibody.
32. A method for determining the presence or amount of the nucleic acid
molecule of
claim 20 in a sample, the method comprising:
(a) providing said sample;

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(b) introducing said sample to a probe that binds to said nucleic acid
molecule;
and
(c) determining the presence or amount of said probe bound to said nucleic
acid
molecule,
thereby determining the presence or amount of the nucleic acid molecule in
said sample.
33. The method of claim 32 wherein presence or amount of the nucleic acid
molecule is
used as a marker for cell or tissue type.
34. The method of claim 33 wherein the cell or tissue type is cancerous.
35. A method for determining the presence of or predisposition to a disease
associated
with altered levels of expression of the nucleic acid molecule of claim 20 in
a first
mammalian subject, the method comprising:
a) measuring the level of expression of the nucleic acid in a sample from the
first mammalian subject; and
b) comparing the level of expression of said nucleic acid in the sample of
step
(a) to the level of expression of the nucleic acid present in a control sample
from a second mammalian subject known not to have or not be predisposed
to, the disease;
wherein an alteration in the level of expression of the nucleic acid in the
first subject as
compared to the control sample indicates the presence of or predisposition to
the disease.
36. A method of producing the polypeptide of claim 1, the method comprising
culturing
a cell under conditions that lead to expression of the polypeptide, wherein
said cell
comprises a vector comprising an isolated nucleic acid molecule comprising a
nucleic acid
sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an
integer
between 1 and 606.
37. The method of claim 36 wherein the cell is a bacterial cell.
38. The method of claim 36 wherein the cell is an insect cell.
39. The method of claim 36 wherein the cell is a yeast cell.
40. The method of claim 36 wherein the cell is a mammalian cell.
41. A method of producing the polypeptide of claim 2, the method comprising
culturing
a cell under conditions that lead to expression of the polypeptide, wherein
said cell
comprises a vector comprising an isolated nucleic acid molecule comprising a
nucleic acid
sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an
integer
between 1 and 606.
42. The method of claim 41 wherein the cell is a bacterial cell.
43. The method of claim 41 wherein the cell is an insect cell.
44. The method of claim 41 wherein the cell is a yeast cell.
45. The method of claim 41 wherein the cell is a mammalian cell.

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Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 9
CONTENANT LES PAGES 1 A 199
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 9
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NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:



CA 02488547 2004-12-02
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THERAPEUTIC POLYPEPTIDES,~NUCLEIC ACIDS ENCODING
SAME, AND METHODS OF USE
FIELD OF THE INVENTION
The present invention relates to novel polypeptides, and the nucleic acids
encoding
them, having properties related to stimulation of biochemical or physiological
responses in
a cell, a tissue, an organ or an organism. More particularly, the novel
polypeptides are gene
products of novel genes, or are specified biologically active fragments or
derivatives
thereof. Methods of use encompass diagnostic and prognostic assay procedures
as well as
methods of treating diverse pathological conditions.



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BACKGROITND OF THE INVENTION
Eukaryotic cells are characterized by biochemical and physiological processes
which under normal conditions are exquisitely balanced to achieve the
preservation and
propagation of the cells. When such cells are components of multicellular
organisms such
as vertebrates, or more particularly organisms such as mammals, the regulation
of the
biochemical and physiological processes involves intricate signaling pathways.
Frequently,
such signaling pathways involve extracellular signaling proteins, cellular
receptors that
bind the signaling proteins, and signal transducing components located within
the cells.
Signaling proteins may be classified as endocrine effectors, paracrine
effectors or
autocrine effectors. Endocrine effectors are signaling molecules secreted by a
given organ
into the circulatory system, which are then transported to a distant target
organ or tissue.
The target cells include the receptors for the endocrine effector, and when
the endocrine
effector binds, a signaling cascade is induced. Paracrine effectors involve
secreting cells
and receptor cells in close proximity to each other, for example two different
classes of
cells in the same tissue or organ. One class of cells secretes the paracrine
effector, which
then reaches the second class of cells, for example by diffusion through the
extracellular
fluid. The second class of cells contains the receptors for the paracrine
effector; binding of
the effector results in induction of the signaling cascade that elicits the
corresponding
biochemical or physiological effect. Autocrine effectors are highly analogous
to paracrine
effectors, except that the same cell type that secretes the autocrine effector
also contains the
receptor. Thus the autocrine effector binds to receptors on the same cell, or
on identical
neighboring cells. The binding process then elicits the characteristic
biochemical or
physiological effect.
Signaling processes may elicit a variety of effects on cells and tissues
including by
way of nonlimiting example induction of cell or tissue proliferation,
suppression of growth
or proliferation, induction of differentiation or maturation of a cell or
tissue, and
suppression of differentiation or maturation of a cell or tissue.
Many pathological conditions involve dysregulation of expression of important
effector proteins. In certain classes of pathologies the dysregulation is
manifested as
diminished or suppressed level of synthesis and secretion of protein
effectors. In other
classes of pathologies the dysregulation is manifested as increased or up-
regulated level of
synthesis and secretion of protein effectors. In a clinical setting a subject
may be suspected
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of suffering from a condition brought on by altered or mis-regulated levels of
a protein
effector of interest. Therefore there is a need to assay for the level of the
protein effector
of interest in a biological sample from such a subject, and to compare the
level with that
characteristic of a nonpathological condition. There also is a need to provide
the protein
effector as a product of manufacture. Administration of the effector to a
subject in need
thereof is useful in treatment of the pathological condition. Accordingly,
there is a need for
a method of treatment of a pathological condition brought on by a diminished
or suppressed
levels of the protein effector of interest. In addition, there is a need for a
method of
treatment of a pathological condition brought on by a increased or up-
regulated levels of
the protein effector of interest.
Antibodies are multichain proteins that bind specifically to a given antigen,
and
bind poorly, or not at all, to substances deemed not to be cognate antigens.
Antibodies are
comprised of two short chains termed light chains and two long chains termed
heavy
chains. These chains are constituted of immunoglobulin domains, of which
generally there
are two classes: one variable domain per chain, one constant domain in light
chains, and
three or more constant domains in heavy chains. The antigen-specific portion
of the
immunoglobulin molecules resides in the variable domains; the variable domains
of one
light chain and one heavy chain associate with each other to generate the
antigen-binding
moiety. Antibodies that bind immunospecifically to a cognate or target antigen
bind with
high affinities. Accordingly, they are useful in assaying specifically for the
presence of the
antigen in a sample. In addition, they have the potential of inactivating the
activity of the
antigen.
Therefore there is a need to assay for the level of a protein effector of
interest in a
biological sample from such a subject, and to compare this level with that
characteristic of
a nonpathological condition. In particular, there is a need for such an assay
based on the
use of an antibody that binds immunospecifically to the antigen. There further
is a need to
inhibit the activity of the protein effector in cases where a pathological
condition arises
from elevated or excessive levels of the effector based on the use of an
antibody that binds
immunospecifically to the effector. Thus, there is a need for the antibody as
a product of
manufacture. There further is a need for a method of treatment of a
pathological condition
brought on by an elevated or excessive level of the protein effector of
interest based on
administering the antibody to the subject.
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SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of isolated polypeptides
including
amino acid sequences selected from mature forms of the amino acid sequences
selected
from the group consisting of SEQ >D NO:2n, wherein n is an integer between 1
and 606.
The novel nucleic acids and polypeptides are referred to herein as NOV 1 a,
NOV lb,
NOVlc, NOVld, NOV2a, NOV2b, NOV2c, NOV2d, NOV3a, NOV3b, etc. These nucleic
acids and polypeptides, as well as derivatives, homologs, analogs and
fragments thereof,
will hereinafter be collectively designated as "NOVX" nucleic acid or
polypeptide
sequences.
The invention also is based in part upon variants of a mature form of the
amino acid
sequence selected from the group consisting of SEQ m N0:2n, wherein n is an
integer
between l and 606, wherein any amino acid in the mature form is changed to a
different
amino acid, provided that no more than 15% of the amino acid residues in the
sequence of
the mature form are so changed. In another embodiment, the invention includes
the amino
acid sequences selected from the group consisting of SEQ ID N0:2n, wherein n
is an
integer between I and 606. In another embodiment, the invention also comprises
variants
of the amino acid sequence selected from the group consisting of SEQ ID N0:2n,
wherein
n is an integer between 1 and 606 wherein any amino acid specified in the
chosen sequence
is changed to a different amino acid, provided that no more than 15°fo
of the amino acid
residues in the sequence are so changed. The invention also involves fragments
of any of
the mature forms of the amino acid sequences selected from the group
consisting of SEQ
ID NO:2n, wherein n is an integer between 1 and 606, or any other amino acid
sequence
selected from this group. The invention also comprises fragments from these
groups in
which up to 15% of the residues are changed.
In another embodiment, the invention encompasses polypeptides that are
naturally
accurnng allelic variants of the sequence selected from the group consisting
of SEQ ID
NO:2n, wherein n is an integer between l and 606. These allelic variants
include amino
acid sequences that are the translations of nucleic acid sequences differing
by a single
nucleotide from nucleic acid sequences selected from the group consisting of
SEQ ID
NOS: 2n-l, wherein n is an integer between I and 606. The variant polypeptide
where any
amino acid changed in the chosen sequence is changed to provide a conservative
substitution.
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In another embodiment, the invention comprises a pharmaceutical composition
involving a polypeptide with an amino acid sequence selected from the group
consisting of
SEQ TD N0:2n, wherein n is an integer between 1 and 606 and a pharmaceutically
acceptable carrier. In another embodiment, the invention involves a kit,
including, in one or
more containers, this pharmaceutical composition.
In another embodiment, the invention includes the use of a therapeutic in the
manufacture of a medicament for treating a syndrome associated with a human
disease, the
disease being selected from a pathology associated with a polypeptide with an
amino acid
sequence selected from the group consisting of SEQ >D N0:2n, wherein n is an
integer
between l and 606 wherein said therapeutic is the polypeptide selected from
this group.
In another embodiment, the invention comprises a method for determining the
presence or amount of a polypeptide with an amino acid sequence selected from
the group
consisting of SEQ >D N0:2n, wherein n is an integer between 1 and 606 in a
sample, the
method involving providing the sample; introducing the sample to an antibody
that binds
irnmunospecifically to the polypeptide; and determining the presence or amount
of
antibody bound to the polypeptide, thereby determining the presence or amount
of
polypeptide in the sample.
In another embodiment, the invention includes a method for determining the
presence of or predisposition to a disease associated with altered levels of a
polypeptide
with an amino acid sequence selected from the group consisting of SEQ ID
N0:2n,
wherein n is an integer between 1 and 606 in a first mammalian subject, the
method
involving measuring the level of expression of the polypeptide in a sample
from the first
mammalian subject; and comparing the amount of the polypeptide in this sample
to the
amount of the polypeptide present in a control sample from a second mammalian
subject
known not to have, or not to be predisposed to, the disease, wherein an
alteration in the
expression level of the polypeptide in the first subject as compared to the
control sample
indicates the presence of or predisposition to the disease.
In another embodiment, the invention involves a method of identifying an agent
that
binds to a polypeptide with an amino acid sequence selected from the group
consisting of
SEQ >D N0:2n, wherein n is an integer between 1 and 606, the method including
introducing the polypeptide to the agent; and determining whether the agent
binds to the
polypeptide. The agent could be a cellular receptor or a downstream effector.
In another embodiment, the invention involves a method for identifying a
potential
therapeutic agent for use in treatment of a pathology, wherein the pathology
is related to
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aberrant expression or aberrant physiological interactions of a polypeptide
with an amino
acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is
an integer
between 1 and 606, the method including providing a cell expressing the
polypeptide of the
invention and having a property or function ascribable to the polypeptide;
contacting the
cell with a composition comprising a candidate substance; and determining
whether the
substance alters the property or function ascribable to the polypeptide;
whereby, if an
alteration observed in the presence of the substance is not observed when the
cell is
contacted with a composition devoid of the substance, the substance is
identified as a
potential therapeutic agent.
In another embodiment, the invention involves a method for screening for a
modulator of activity or of latency or predisposition to a pathology
associated with a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID
NO:2n, wherein n is an integer between l and 606, the method including
administering a
test compound to a test animal at increased risk for a pathology associated
with the
polypeptide of the invention, wherein the test animal recombinantly expresses
the
polypeptide of the invention; measuring the activity of the polypeptide in the
test animal
after administering the test compound; and comparing the activity of the
protein in the test
animal with the activity of the polypeptide in a control animal not
administered the
polypeptide, wherein a change in the activity of the polypeptide in the test
animal relative
to the control animal indicates the test compound is a modulator of latency
of, or
predisposition to, a pathology associated with the polypeptide of the
invention. The
recombinant test animal could express a test protein transgene or express the
transgene
under the control of a promoter at an increased level relative to a wild-type
test animal The
promoter may or may not b the native gene promoter of the transgene.
In another embodiment, the invention involves a method for modulating the
activity
of a polypeptide with an amino acid sequence selected from the group
consisting of SEQ
ID N0:2n, wherein n is an integer between 1 and 606, the method including
introducing a
cell sample expressing the polypeptide with a compound that binds to the
polypeptide in an
amount sufficient to modulate the activity of the polypeptide.
In another embodiment, the invention involves a method of treating or
preventing a
pathology associated with a polypeptide with an amino acid sequence selected
from the
group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 606,
the method
including administering the polypeptide to a subject in which such treatment
or prevention
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is desired in an amount sufficient to treat or prevent the pathology in the
subject. The
subject could be human.
In another embodiment, the invention involves a method of treating a
pathological
state in a mammal, the method including adminustering to the mammal a
polypeptide in an
amount that is sufficient to alleviate the pathological state, wherein the
polypeptide is a
polypeptide having an amino acid sequence at least 95% identical to a
polypeptide having
the amino acid sequence selected from the group consisting of SEQ ID N0:2n,
wherein n is
an integer between 1 and 606 or a biologically active fragment thereof.
In another embodiment, the invention involves an isolated nucleic acid
molecule
comprising a nucleic acid sequence encoding a polypeptide having an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given SEQ
>D N0:2n, wherein n is an integer between 1 and 606; a variant of a mature
form of the
amino acid sequence selected from the group consisting of SEQ 1D N0:2n,
wherein n is an
integer between l and 606 wherein any amino acid in the mature form of the
chosen
sequence is changed to a different amino acid, provided that no more than 15%
of the
amino acid residues in the sequence of the mature form are so changed; the
amino acid
sequence selected from the group consisting of SEQ 1D N0:2n, wherein n is an
integer
between 1 and 606; a variant of the amino acid sequence selected from the
group consisting
of SEQ m N0:2n, wherein n is an integer between 1 and 606, in which any amino
acid
specified in the chosen sequence is changed to a different amino acid,
provided that no
more than 1 S% of the amino acid residues in the sequence are so changed; a
nucleic acid
fragment encoding at least a portion of a polypeptide comprising the amino
acid sequence
selected from the group consisting of SEQ )D N0:2n, wherein n is an integer
between 1
and 606 or any variant of the polypeptide wherein any amino acid of the chosen
sequence is
changed to a different amino acid, provided that no more than 10% of the amino
acid
residues in the sequence are so changed; and the complement of any of the
nucleic acid
molecules.
In another embodiment, the invention comprises an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide comprising an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given SEQ
1D N0:2n, wherein n is an integer between 1 and 606, wherein the nucleic acid
molecule
comprises the nucleotide sequence of a naturally occurnng allelic nucleic acid
variant.
In another embodiment, the invention involves an isolated nucleic acid
molecule
including a nucleic acid sequence encoding a polypeptide having an amino acid
sequence
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selected from the group consisting of a mature form of the amino acid sequence
given SEQ
ID N0:2n, wherein n is an integer between 1 and 606 that encodes a variant
polypeptide,
wherein the variant polypeptide has the polypeptide sequence of a naturally
occurring
polypeptide variant.
In another embodiment, the invention comprises an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide comprising an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given SEQ
ID N0:2n, wherein n is an integer between 1 and 606, wherein the nucleic acid
molecule
differs by a single nucleotide from a nucleic acid sequence selected from the
group
consisting of SEQ ID NOS: 2n-1, wherein n is an integer between 1 and 606.
In another embodiment, the invention includes an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide including an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given SEQ
ID N0:2n, wherein n is an integer between 1 and 606, wherein the nucleic acid
molecule
comprises a nucleotide sequence selected from the group consisting of the
nucleotide
sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an
integer
between 1 and 606; a nucleotide sequence wherein one or more nucleotides in
the
nucleotide sequence selected from the group consisting of SEQ 1D N0:2n-1,
wherein n is
an integer between l and 606 is changed from that selected from the group
consisting of the
chosen sequence to a different nucleotide provided that no more than
15°!° of the
nucleotides are so changed; a nucleic acid fragment of the sequence selected
from the
group consisting of SEQ lD NO:2n-1, wherein n is an integer between 1 and 606;
and a
nucleic acid fragment wherein one or more nucleotides in the nucleotide
sequence selected
from the group consisting of SEQ ID N0:2n-l, wherein n is an integer between l
and 606
is changed from that selected from the group consisting of the chosen sequence
to a
different nucleotide provided that no more than 15°!° of the
nucleotides are so changed.
In another embodiment, the invention includes an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide including an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given SEQ
ID N0:2n, wherein n is an integer between 1 and 606, wherein the nucleic acid
molecule
hybridizes under stringent conditions to the nucleotide sequence selected from
the group
consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 606, or a
complement of the nucleotide sequence.



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
In another embodiment, the invention includes an isolated nucleic acid
molecule
having a nucleic acid sequence encoding a polypeptide including an amino acid
sequence
selected from the group consisting of a mature form of the amino acid sequence
given SEQ
m N0:2n, wherein n is an integer between 1 and 606, wherein the nucleic acid
molecule
has a nucleotide sequence in which any nucleotide specified in the coding
sequence of the
chosen nucleotide sequence is changed from that selected from the group
consisting of the
chosen sequence to a different nucleotide provided that no more than 15% of
the
nucleotides in the chosen coding sequence are so changed, an isolated second
polynucleotide that is a complement of the first polynucleotide, or a fragment
of any of
them.
In another embodiment, the invention includes a vector involving the nucleic
acid
molecule having a nucleic acid sequence encoding a polypeptide including an
amino acid
sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ ID N0:2n, wherein n is an integer between 1 and 606. This vector can
have a
promoter operably linked to the nucleic acid molecule. This vector can be
located within a
cell.
In another embodiment, the invention involves a method for determining the
presence or amount of a nucleic acid molecule having a nucleic acid sequence
encoding a
polypeptide including an amino acid sequence selected from the group
consisting of a
mature form of the amino acid sequence given SEQ )D N0:2n, wherein n is an
integer
between l and 606 in a sample, the method including providing the sample;
introducing the
sample to a probe that binds to the nucleic acid molecule; and determining the
presence or
amount of the probe bound to the nucleic acid molecule, thereby determining
the presence
or amount of the nucleic acid molecule in the sample. The presence or amount
of the
nucleic acid molecule is used as a marker for cell or tissue type. The cell
type can be
cancerous.
In another embodiment, the invention involves a method for determining the
presence of or predisposition for a disease associated with altered levels of
a nucleic acid
molecule having a nucleic acid sequence encoding a polypeptide including an
amino acid
sequence selected from the group consisting of a mature form of the amino acid
sequence
given SEQ m NO:2n, wherein n is an integer between l and 606 in a first
mammalian
subject, the method including measuring the amount of the nucleic acid in a
sample from
the first mammalian subject; and comparing the amount of the nucleic acid in
the sample of
step (a) to the amount of the nucleic acid present in a control sample from a
second
11



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
mammalian subject known not to have or not be predisposed to, the disease;
wherein an
alteration in the level of the nucleic acid in the first subject as compared
to the control
sample indicates the presence of or predisposition to the disease.
The invention further provides an antibody that binds immunospecifically to a
NOVX polypeptide. The NOVX antibody may be monoclonal, humanized, or a fully
human antibody. Preferably, the antibody has a dissociation constant for the
binding of the
NOVX polypeptide to the antibody less than 1 x 10-9 M. More preferably, the
NOVX
antibody neutralizes the activity of the NOVX polypeptide.
In a further aspect, the invention provides for the use of a therapeutic in
the
manufacture of a medicament for treating a syndrome associated with a human
disease,
associated with a NOVX polypeptide. Preferably the therapeutic is a NOVX
antibody.
In yet a further aspect, the invention provides a method of treating or
preventing a
NOVX-associated disorder, a method of treating a pathological state in a
mammal, and a
method of treating or preventing a pathology associated with a polypeptide by
administering a NOVX antibody to a subject in an amount sufficient to treat or
prevent the
disorder.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention, suitable
methods and materials are described below. All publications, patent
applications, patents,
and other references mentioned herein are incorporated by reference in their
entirety. In
the case of conflict, the present specification, including definitions, will
control. In
addition, the materials, methods, and examples are illustrative only and are
not intended to
be limiting.
Other features and advantages of the invention will be apparent from the
following
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded
thereby. Included in the invention are the novel nucleic acid sequences, their
encoded
22



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
polypeptides, antibodies, and other related compounds. The sequences are
collectively
referred to herein as "NOVX nucleic acids" or "NOVX polynucleotides" and the
corresponding encoded polypeptides are referred to as "NOVX polypeptides" or
"NOVX
proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the
novel
sequences disclosed herein. Table A provides a summary of the NOVX nucleic
acids and
their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers
NOVX Internal SEQ ID SEQ ID Homology
Assignment ' Identification NO NO
(nucleic ~ (amino
1a tCG191083-O1 g~~ ,~ON WILLEBR.AND factor A-
,,[related protein homolog - Mus
CG191745-01 f,4~ ntegrin alpha-2 precursor
3 ~ (Platelet
' I


OV2a , membrane glycoprotein
Ia) (GPIs)


~( CD49b) - Homo sapiens


3a 253-O1 5 ~, Human secreted protein
OV ~ CG50 6 SCEP-39
~ ~ _ _ _ _ _.
~


_ _ . ~y Human secreted protein_SCEP-39
_ __ ~~
_ .
fOV3b~7
- :250095765
~


_- , ~ 10 Human secreted protein
tOV3cm~250095742 9 SCEP-39


TOV3d 250095779 ! ~' Human secreted protein
11 4 ~ 12 SCEP-39


dOV3e '250095794 14 Human secreted protein
13 ' SCEP-39


JOV3f 15 _ Human secreted protein
250095734 ~ SCEP-39
~ 16
~4
Y


JOV3g _ 18 Human secreted protein
250095811 17 SCEP-39


3h ' 19 20 Human secreted protein
~ ~~250095799 SCEP-39
JOV


_ _ 22 Human secreted protein
~10V3i~~SNP13377609 SCEP-39
21


~10V3j929 23 24 Human secreted protein
3 SCEP-39
7
133
SNP


k ~ _ 25 ~ 26 Human secreted protein
~TOV3 _ ~ SCEP_-39
_ 4
_
'SNP13380272


_ ' SNP13379745 27 28 Human secreted protein
~OV31 SCEP-39
~
~


~10V3m3373930 29 30 Human secreted protein
SNPl SCEP-39
~


NOV4a _ 31 32 C_SMD1 - Mus musculus
04
' CG50377-


_ _ -
NOV4b _ ,~..-~ 34 ~~CSMD1 - Mus musculus
~ ~ CG50377-O133


NOV4c ' CG50377-02 35 36 CSMD1 - Mus musculus


NOV4d ' CG50377-03 37 38 ~ CSMD1 - Mus musculus
n


NOV4e ' CG50377-OS 39 40 CSMDl - Mus musculus
~ ~


~NOV4f~ CG50377-06 41 .42_ CSM_D1 - Mus musculus
~
~


NOV4g ' 273095147 4 44 ., CSMD 1 _~Mus musculus
X 3 ~


NOV4h ' 317459653 _ 46 CSMDl - Mus musculus
45


NOV4i ' 317459612 47 ~ 48 CSMD1 - Mus musculus
_
_


13



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV4j 317286331 49 50
___.._.......
___ _ ..... _ ._; .. ..... ~ CS
_ . . . ... _ _... . . _.. MD1 - M
__.. ....._. . us m
. ... _._ usculus
. ..


.. ._ _.. _...._
NOV4k ~ CG50377-07.._._.. _
_._.. ._.: __.........SI 52 _
..__..__ ... . ._ ___ _... ....._..._ . _... .. _.. ......
.. .. . . .........._......._ _ .. ._.. _... ............._........
.... ....__. . _... ..._ _.......
. . ~t CSMD1 - Mus musculus
~


. . _"~.....__,__._...
NOV_41 CG50377-08 53 _.. ...~"~,~x_. __......_...
___ _ _ .._..........__._.__.,.
.~_ .. _ ...._..........
CSMD1 - Mus musculus


_
NOV4m CG50377-09 55 56 CSMD1 -
~ ~ ~ Mus musculus


NOV4n CG50377-10 57 _
~ 58 ~ CSMD1
- Mus musculus


NOV4o CG50377-11 :59 60 _
y CSM
~ Dl ~- Mus musculus


NOV4p SNP1338245761 _
62 CSMD1 - Mus musculus
Y
n


__
NOV4q SNP1338245863 64 CSMDl -MusmusculusY
-


NOVSa :CG50389-045 ~Interleukin 1 receptor-like
6 - 66 2


'precursor (IL-lRrp2)
(IL1R-rp2) -
'


__ - _ Homo
Y sa iens
~ p


_
NOVSb ~CG50389-0667 __
X68 Interleukin 1 receptor-like
~ 2


'precursor (IL-lRrp2)
(IL1R-rp2) -


jHomo Sapiens
4y d ~


NOVS c :25744864869 70 Interleukin 1 receptor-like
2


precursor (IL-lRrp2)
(IL1R-rp2) -


_ ~_ _ . Homo sapien
- s


NOVSd CG50389-Op 71 _
72 ~Interleukin 1 receptor-like
2


precursor (IL-lRrp2)
(IL1R-rp2) -


Homo sapiens
~


~VOVSe CG50389-02 73 74 Interleukin preceptor-like
~ 2


precursor (IL-lRrp2)
(IL1R-rp2) -


Homo Sapiens
.


VOVSf CG50389-03 75 Interleukin 1 receptor-like
~ 76 2


precursor (IL-lRrp2)
(IL1R-rp2) -


a Homo sa iens
p


~OVSg CG50389-OS 77 78 Interleukin 1 receptor-like
~ 2


precursor (IL-lRrp2)
(IL1R-rp2) -


Homo Sapiens


JOVSh SNP1338246479 80 Interleukin 1 receptor-like2


precursor (IL-lRrp2)
(IL1R-rp2) -


Homo Sapiens


TOV6a CG50391-08 81 82 Human protease-inhibitor
like


_ .. .p rotein
..
~


lOV6 b _ . Human protease-inhibitor
, CG50391-09 .... . like
8 3 84


_ _ ~ j ~p ro
~ ~ tein


fOV6c CG50391-10 5 86 _
: 8 Human protease-inhibitor
like


~ ~ p rotein


_
OV6d C G50391-Ol 7 88 'H uman protease-inhibitor
8 like


p rotein


OV6e C G50391-02 9 90,H uman protease-inhibitor
8 like


._._.. .__ _....__._..............~~_ __~ rotein
_..; . . _ .............._.
. . ._.. ~
~p


~ ...._. _. _.._...._.-....
OV6f C G50391-03 . .... . .. ... _ _
9 . _~__.._...._..uman protease-inhibitor
.u,~. like ~ ~~
1 92 H


p rotein


OV6g C G50391-04 3 94 -Y- H uman protease-inhibitor
9 like


14



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
protein
_........_. . _.. ___......_... _.. ... _
.._..__..._.._._......._...._.._._.._............._........._..,__.___._.__._..
._...
_ ....._... _.... ... .._ . . ... ...
........ _._..__ ..__.._......
. _. . _ ... _. ....... _.
.._._.. .


NOV6h , , Human protease-inhibitor
_.._...._ 5 95 96 like
_ ........_.....~05 ..... ._._. protein _. .. ...........
..._. CG 0391 _ . ..._ ... _ _. .... ..... . ......
. :. .. ...._..._..... . . _._ _... . .._. . .... _._ . ..
.... _.._. .... . . _.. . ._. . ... ..
.._ .. .. _.~
. _ .


NOV6i CG50391-06 97 ' 98 Human protease-inhibitor
~ like
NOV6j -_- _ 99 100 protein
CG50391-07 Human protease-inhibitor
NOV6k 141 102 like
_._..v_ CG50391-11 _ ~ _._ protein
_..~.. ___ __ __ Human protease-inhibitor
. like
protein - _ . ._._. __


NOV61 SNP13376336103 ' 104 Human protease-inhibitor
like
_. ._... __ _ _....._..........._............protei
.... ....._.SNP13376335. ......, ...... ___..n...._.. _.._
_.. _ ~_____ 105 106 ... ..... ..... _._....._
NOV6m ___ ___._.. .. ...._.... .....__.......
_ _ _ _ __ ~_ _...
___.. Human protease-inhibitor
_ like
protein
______ ____~ __ ,__.__
_ ,.._ __ ~_ _ ~ _~_


NOV6n SNP13376334107 108 Human protease-inhibitor
: _ _ _ like
NOV6o 109 -~ ~ 110 protein
_ SNP13376333~ __ . ~~ Human protease-inhibitor
_ like
protein~ ~ -_~ _ _


NOV6p SNP13382488111 112 Human protease-inhibitor
: 3~ like
protein..... . ....
~


NOV7a CG50426-17 113 114 Ten-m2 - Mus musculus
_ _........ .. ._._. _.
_......._ .. ....... . _.... _..
...._.._._........_........._......_ ._ ...
_... .........


NOV7b : 115 Ten m2 Mus musculus
~ CG50426-21 116 ..........._....._..............
..............................__ _......... _...... ...........
....... . ..._. ............_............. . _...........
......... a........ ............. ..... . ..........
.... _........_................... ...... .. .....
........ .... .......
.


NOV7c CG50426-15 117 118 Ten-m2 - Mus musculus
? f ..
............................................,......,..,."....",_._,.,_......~..
., ..... .,............. ..........._._......................
.. ....................................._.3 _ ........_._ , .....
....._ .. _........,_.
.._. .... . ......._.... _ .......
....._........ .............
_._._.,.....


NOV7d ' 306276924 , Ten-m2 - Mus musculus
; 119 120 ' _. _. .. _ ....
_.....__....__. _.. . .. . _.. . W... __...
_..._._..................._..._ . ._... .. _. ...... ._ .__.
~ ~
~~


_ ' 121 Ten
NOV7e 306276936 122 _
~ x ~ ,.. ...,.........._. , -m2 - Mus musculus
. ,.__. ..
_...


NOV7f 308530526 123 124 Ten-m2 - Mus musculus
. .. ......................................_.._. .. .. ...__.._...._.
...._......._........
_........ .. _ _......_..._..._ ._ _..._
_.._.__.._...._..__.........._.._.._..
.................._. .. . _....._._. _..................._.........


NOV7g 308530589 125 126 Ten-m2 -Mus musculus


NOV7h CG50426-Ol 127 128 Ten-m2 Mus musculus
_... . . _.. _ . . _ _ _ _ .
._. _. CG50426-02 129 . .... .......Ten-~_-..Mus musculus
_ _ _. _~ 130 ...... _._.... . ..... ....
_: . . ..._~
NOV7i ..
.....


NOV7j CG50426-03 131 132 _ Ten-m2 - Mus musculus
~


NOV7k CG50426-04 133 l 34 Ten-m2 - Mus musculus
~ . ...... .. . ..
.... . ~
. .. ... .._ _
.


NOV71 CG50426-05 135 Ten-m2 - Mus mu_sc_ul_us
... . . .. . 136 _ ..
# ~.. .= y
_..,~_ .........
~
~


NOV7m ~G50426-06 13$ Ten-m2
_.........___.__... 137 -Mus musculus
...._.._. _. _ ,~ _._..._ . _.._ ._ ._.__.
_. . . _ .. ._ _.._.. _.... ....._..._...._..
_
..._._ __
~..


NOV7n ~ 3 ~ ~
CG50426 ! 140 ~ -
07 139 Te
_ . _ .... ... . . . _ n m2 Mus musculus
_ _. . _._._.. .._ .._ .. ..
. ... . .._. ... ...
._ . .. . . . .


NOV7o CG50426-08 141 142 Ten-m2 - Mus musculus
_........
........_......................................................................
.._..._.._......................._.....__._.................._.............__..
....._......
..............._.....: __.....~


NOV7p CG50426-09 143 144 Ten-m2 -Mus musculus
_....._ .- ...... ...... ............ _ ..__ . __ _ _ __.._.
_ . ._._........___... _... _..._ _... _.._... ...... ..._.
_.._._ . _.__ . ._._.. ..__.... . .._....
..__ . . _.___


NOV7q CG50 145 146 Ten-m2 - Mus musculus
_... _ 426-10 ..._...... ._ ........__.__ _ ..._.
.. _ _ .. .__. . ..._....__..._. . .._. . ..... . _
NOV7r ...._ ... _.... . ......_.. . _ _ _ .. _._...._.
_._ . _......_ . ...._.._.. ..
._..__ CG50426 ... Ten m2 Mus musculus
11 147 148 . . . ..
.. . .._..... ..._.
__._..
.
. .


.. CG50426-12 ~ Ten-m2 - Mus musculus
; 50
NOV7s i
a 149
'


NOV7t CG50426-13 151 152 Ten-m2 - Mus musculus
' ._._._. _ ..................._,_......._._...a._....,.._..._.
..._,............._._......_._...,__..___.._...__.....
.... _........_ .,._.._.__..__......,......_.............__.......~
..... ~,~,;___._.,.,..__._,.,..,.._._
....._...,_.._._..__...,-;,._ .._,......._..


NOV7u CG50426-14 , ,
_.. 153 i 154 ,
_. _ _ . .... Ten m2 Mus musculus
_.. . __.__._............
. _.. . _ .._ .. .. _


NOV7v . Ten-m2 - Mus musculus
._........CG50426 155 __. _....._._. .._ _._. ..
_... _. 16 - . .. _.... ...._ _.. . .... .. _..
..... .. . ... ... . _. . .. .._..__
....... .. ..... ... 156 ~
_........
...' _..............


NOV7w CG50426-18 157 158 Ten-m2 - Mus musculus
........ ~ .._......_;n ..._. .. c ......... .. _. .. .
._ ..._ ... . ....... .... ...... .. ... _..._........
.. ... ...._.. .... .. .._ . .. ... .. . . ....
. ._.. .... . ..N........ _.. . . .._ .... .. .
_ _.. _ , ........... .... ....._..... .... _ . . . ..
... . ..


NOV7x ~ CG50426-19159 160 ~ Ten-m2 - Mus musculus





CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
11~~ ~!Y _y:ci5U4~6-20161 Ten-m2 -
.._......... 162 u
M l
~ ~


s muscu
NOV7z CG50426-22 ..._.1~.__.__....__.._..~,~. us
~ .. ..-,
_....~.._....._._.._ -......_......._..___...__.._ . _ ....
..._ ... ._.......
_... .. .. _.......63 164 ..._..~__._.__..._...~_._._..._.....
... __..~_.__.~a~.............__........_.... ,__
.., ..... Ten-m2 - Mus musculus
_._..


_ ...... ...; ._...._.. __._.
NOV7a CG50426 ..._. _. _.......-
~_...__...___........._.~"~_.__..._.._....
23 .. Ten
165 -m2 - Mus m
166 usculu
. s


NOV7b ' CG50426-24' 167 _
w ~ 168 _
~ _
~~ -' Ten-m2
- M
us m
uscul
us~.


NOV7c CG5042_6-25169 _
170 _
J _
_
Ten-m2 -Mus mu
scul
us


NOV7d ~SNP13375665171 _
~ 172 _
~~ Ten-m2 - Mus musculus~~~~


NOV7e SNP13375213173 Ten-m2 - Mus musculus
174


NOV7f : SNP 13374985175 ~ Ten-m2 '- Mus musculus
176
y


NOVBa CG50646-04 177 Polydom protein precursor
178~~ - Mus


musculus
....._.... _._
.
..._


NOV8b 237582568 1 .Pol dom.....~ote~.........
79 .. __ ....... .
.........._...._.... ' Y p m precursor -
l..go_.._.._._...._... Mus


~ ~ ~ ~ ~musculus


NOVBc 236 . _ Polydom protein precursor
; 589434 _ - Mus
181
182


musculus


~._
NOVBd 236495259 ;183 Polydom protein precursor
-184 - Mus


~musculus__
NOVB : ~ ~
~. ~


e 236495256 ;185 Polydom
~ ~ 186 protein precursor -
Mus


__. ....._..... ..... ' ......_.. musculus
........ . _.._ .._
. ........_......
_ ..... .
. ..._
..... ..


._ ... _ . . . .. . ... .
NOV8f CG50646-Ol . . ._..__. _... .._..
_ ...._. _... .. ...
. .... .. ._... . . ....._...
. _ . ... .
187 'Polydom protein precursor
188 - Mus


~musculus


_
NOVBg CG50646-02 189 Polydom protein precursor
~ 190 - Mus
,


~musculus


LVOVBh CG50646-03 191 Polydom protein precursor
192 - Mus


musculus
~


NOV8i CG50646-OS 193 Polydom protein~precursor
~ 194 - Mus


. ._.. musculus
~ ._....
_
~
~


. . . .... .... _._ ....__...
VOVBj SNP 13380898._ .... ...
.. .._ Polydom protein precursor
195 _.. - Mus
_.....__
_
.
196


~ cuIus
mus


~OVBk SNP13380899197 ~ 198 y
- ~ p
~___
Pol dom rotein precursor
- Mus


musculus


JOV81 SNP13374702199 200 Polydom protein precursor
- Mus


~ musculus


JOV8m SNP13374257201 202 ~~ Polydom protein precursor
- Mus


. . _ .._ ' musculus
~ _ . _ . .__ L
_. ~. _. ~


,_.~:~ . ... . . _ _.._..._... ..
dOVBn NP13382479 _ . _... _.._.. ___ _ _
S ,2 03 .._ olydom protein precursor
i _........-~Mus~
204 P


musculus


_
lOVBo NP13382480 05 206 P olydom protein precursor
' S 2 - Mus


~m usculus


fOV9a G50736-09 07 208 C D44-Like precursor
~ C 2 FELL - Homo


~s ap
~ -- . ~_ iens
~


OV9b 1 97408749 09 210 C _
~ 2 _
D44-Like


precursor FELL - Homo
~


_.._.. ..... ; S apiens
_..
. ._.
_._


OV9c C G50736-01 .... _ _. D44-like precursor
2 .._. . _ FELL - Homo
_ 212 C
11


16



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV9d ? CG50736-02213 214 ' CD44-like precursor
FELL - Homo


_ __ _ ~, __ _ sapiens


NOV9e CG50736-03 215 ~ 216 i CD44-like precursor
FELL - Homo


sapiens
a J


NOV9f CG50736-04 217 CD44-like precursor FELL
218 - Homo


sapiens


NOV9g CG50736-OS 219 220 ~ CD44-like precursor
FELL - Homo


_ Sapiens


NOV9h CG50736-06 221 222 CD44-like precursor FELL
- Homo 4


sapiens
.............._... .................... .......... .. ..................._.
__ .... _ . .._.... ....
..... .... _.
. ..... ._...........


NOV9i ; . -
_...._.....W................_................_.._..._......___._..~..,~,
CG50736 223 224 ~CD44-like precursor
07 FELL - Homo


__ sapiens


NOV9j ~ CG50736-08 225 226 ~ CD44-like precursor FELL
- Homo


Sapiens


NOV9k ' CG50736-10 227 228 CD44-like precursor FELL
- Homo


_ Sapiens
-


NOV91 a CG50736-11 229 230 CD44-like precursor FELL
- Homo


_......._...._._. .. ...._.... . ....._. S~piens ... __.
_...; .........._...........__ .... .... .
.... . _ .... .. ..._....
....... . ._.... ....
..~


..
NOV9m CG50736-12 231 232 , . ....._... . ...........
.... .................
_......
CD44-like precursor FELL
- Homo


- _ _ _ I sa_piens
T


NOV9n CG50736-13 233 234 " CD44-like precursor FELL
' - Homo


_ sa
~ Y ~ piens
~


NOV9o CG50736-l 235 _
' 4 ~ 236~~ CD44-like~.precursor
FELL - Homo


_ ~., E s sap
iens


NOV9p CG50736-15 237 238 _
CD44-like precursor FELL-
Homo


~ sapiens
~..........._._..__.... ..............._...._....._..._......._....._.
_..._._.. _ _.......
..


NOV9 ...
q : CG50736 239 240 . _.._.._.._......_..........
16 .._..._..... ............
._..
CD44-like precursor FELL
- Homo


_ ~ sapiens


NOV9r CG50736-17 241 242 CD44-like precursor FELL
- Homo


Sapiens
.


NOV9s CG50736-18 243 CD44-like precursor FELL
244 . - Homo


3
Sapiens


NOV9t CG50736-19 245 246 CD44-like precursor FELL
- Homo


_ ~ ~ s_apiens
~~ y 4 ~u
~~ y ~


NOV9u CG50736-20 247 CD44-like precursor FELL
248 -Homo


_ ~ Sapiens


NOV9v CG50736-21 249 250 CD44-like precursor FELL
- Homo


~ sap
iens


_
NOV9w CG50736-22 251 ' 252 __
~ ~ CD44-like precursor FELL
- Homo


_, s apiens
__ _ . "


_ _
NOV9x CG50736-23 253 254 ~ CD44-like precursor,FELL
- Homo


=S apiens
_.... . .. .._. _......_._.._._ ._ . .
_ . . . ...... _._ _._
_. ... _.. _
.


y ~4 ~ ~
NOV9 CG50736 .25 :
55 6 _
p_........._. .. .. _
. __._ ._. ._. _ ._..
_ . _
CD44 hke recursor FELL
Homo


__ S apiens
___ ~. ,


NOVlOa CG50925-Ol 257 258 Tumor endothelial marker
S


17



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
__ _ -:~ .... !precursor_- H_om_o sapiens
~ _ _ _i
~


_
NOV l Ob CG50925-08 _
259 260 Tumor endothelial marker
5


_ _ ,.. , precursor - Homo sapiens
.. , ...._.
_ . ...
~u


NOV I Oc 226990087 261 262 Tumor endothelial marker
5


precursor - Homo
sap
iens


NOVlOd 320054488 263 264 _
_
Tumor endothelial marker
5


_ precursor - Homo sa iens
P


NOVlOe CG50925-02265 266 Tumor endothelial marker
5


precursor - Homo sapiens
y


NOVlOf 248208 267 268 Tumor endothelial marker
844 5 -


~precursor - Ho
....... ........................._.. _................mo Sapiens
_ _. _....._.. .... . _. _........... ....._
.._......


NOV , _
l Og CG50925 269 270 ..... ... .. _...._.....
03 ~ ...
Tumor endothelial marker
5


__ ___ __~_ . pr_ec_urs_or - Homo_s_ap
~~ _ ~ _ ~_ _ , _~_ iens
~


_
NOV 1 Oh CG50925-04271 272 _ __
Tumor endothelial marker
5


j precursor - Homo Sapiens


NOV l0i CG50925-OS273 274 Tumor endothelial marker
5


_ _ _ _ _. ~- precursor - Homo Sapiens
~ ~


NOV 1 Oj CG50925-06275 276 Tumor endothelial marker
~ 5


_ _. _ . . _.j _.. precursor Homo Sapiens
_,_.
~


_
NOVIOk CG50925-07277 278 Tumor endothelial marker
~ 5


_ _ __ precursor - Homo sapien
~ s


NOVIOI SNP13380040279 280 _
: Tumor endothelial marker
5


_ precursor - Homo sapiens


NOVl la CG51027-06281 282 Human immunoglobulin superfamily


protein IGSFP-9


NOVllb 278391205 283 284 Human
; i mmunoglobulin superfamily


__. . ....... -... __.. ... ..... protein IGSFP-9
. . ................_ ...._. _...._. _ _ _
.. . .. ._ _


_
NOV 1 l 278391050 ~85 ... _. . _....... _..._
c 286 . ..... __....... . ...
~ . ..._. ....... ._._
. .. _..
Human immunoglobulin superfamily


_ _ protein IGSFP-9


NOVl ld 277582210 ~ Human immunoglobulin superfamily
; 287 288


protein IG
SFP
-9


_
NOV 11 277582173 289 290 j _
a __
X Human immunoglobulin superfamily


~ _ _ ~ protein IGSFP-9


NOV 11 CG51027-09291 292 Human immunoglobulin superfamily
f


_.. ..._ .. _~ protein IGSFP-9
.... . ~
. ......._


NOV l l 316763704 293 Human immunoglobulin superfamily
g 294


protein IGSFP-9


NOV1 lh CG51027-Ol295 296 Human immunoglobulin superfamily


protein IGSFP
-9


NOV l l CGS 1027-02297 298 _
i ~ ' Human immunoglobulin superfamily


_ _ - ~ _ ~ pr
~~ ~ ~ otei
n IGS
FP-9


NOVl lj CG51027-03299 _
' '300 j _
_
_
Human immunoglobulin superfamily


__ .. ............ _ ._ .. . .. ._ .._..._._Protein IGSFP-9
_ . _ ... . .....__. ...._..
._.._.._ ..__ _.........
.... _ ...
... _..._.
_


..
NOVllk - 301 302 ' _....._... -i
CG51027 Human immunoglobulin superfamily
04


~ protein IGSFP-9


18



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
~10V111 CG51027-OS X303. _.....";-"._.~ ~._. ~.
~ 304 ..
Human immunoglobulin superfamily


protein IGSFP-9


VOVllm CG51027-07 305 306 Human immunoglobulin superfamilyf


protein IGSFP-9
~


VOV1 CG51027-08 307 308 Human immunoglobulin superfamily
In


protein IGSFP-9


_
~TOV CG51027-10 > 309 ~ Human immunoglobulin~superfamily
110 ; 310


- protein IGSFP-9


JOVllp CG51027-1I 311 312 Human~immunoglobulinsuperfamily


~.-... 3 .. ......._.......... protein
...... . _.... .......... IGSFP-9
_.. .._.._.... .._......
_ . ..~


JOV 11 ~ CG51027-12_.. .~ ,.
q ..~ SHuman immunoglobulin
.. . superfamily
_...
_
313 314


protein IGSFP-9
~ v


JOVllr SNP13376340_ Human immunoglobulin superfamily
315 316


protein IGSFP-9


TOV 11 SNP 13376341317 318 Human immunoglobulin superfamily
s ~


__ protein IGSFP-9
_ _ Y


- _
lOVl2a CG51373-11.319 320 Human G protein-coupled
a ~ . receptor


fOV.l2b ' CG_51373-10321 322 Human G protein-coupled
. . ...,-_. ;3_._ receptor
_ t

w


fOVI2c CG51373-08 323 324 Human G protein-coupled
_ . . _ .n___ receptor
~~


fOVl2d 237582_097_325 326 ~ Hum
_ ~ ~ _.___ an
~ - . ~ Gprot
- ei
n-coupled
recep
tor


OV 12e 2943 85961 327 328 _ _
, y y _
_
_
_
_
Human G protein-coupled
receptor


OV 12f 294398021 329 ~ 330 Human G protein-coupled
~I receptor


OVl2g CG51373-Ol 331 332 Human G protein-coupled
' , . receptor
~
~ ~


OV 12h CGS_1373-02333 334 Human G protein-coupled
, ; receptor
~ _.


OVl2i CG51373-03 335 336 Human G protein-coupled
' Y - receptor -
t


OVl2j CG51373-04 337 338 Human Gprotein-coupledreceptor~
F Y -


OV 12k CGS 1373-OS339 340 Human G protein-coupled
~ y ~ receptor


OV121 _ 341 342 Human G protein-coupled
CG51373-06 receptor
V


~V 12m CGS 1373-07343 _ 3_44 Human G protein-coupled
~ - - receptor


~Vl2n CG5137_3-093_45_ 346_ Human
~ G protein-coupled receptor


~V 120 CGS 1373 347 34_8 _
! I 2 ~ ~,.. ! Human G protein-coupled
. ~ - ~ -~~ receptor


~Vl2p CG51373_-133_49 350 HumanG pr
_ ~ otei
~ n-coupl
e
d~receptor


)V 12q CG51373-1435_1 352 _
~ I _
~ _
_
Human G protein-cocoupl
edrece
ptor
_


. _
)Vl2r NP13377352 53 354 _
S X3 ~ -:~_,
Human Gp
rote
in-
coupled receptor


)Vl2s NP1_337974655 - 356 _
_ S ,3 _~ _
4 - _
HurnanGprotein-coupIedreceptor


)V 12t NP 1337485957 358 H uman G protein-coupled
i S 3 a receptor
4


)Vl2u NP13374858 59 360 H uman G protein-coupled
S 3 receptor


)Vl2v NP13374856 61 362 Human G protein-coupled
S 3 .y ~ receptor
~


)Vl2w NP13374855 63_ 364 _~H uman
_ v 3 G protein-coupled receptor4-
S
l' W


)Vl2x NP13374854_65 366 H _
S 3 umanGprotein-coupled receptor
~
.


>Vl2y NP13374853 67 368 H uman G protein-coupled
_ 3 _ _n. receptor
S


1V 13a G51622-04 69 370 V on Ebner minor protein
C 3 - Homo


19



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV 13b 371 ~ 372 Von Ebner minor protein
CG51622-03 - Homo


__ _ _ ~ sapiens
' n


NOV 13c y 373 374 Von Ebner minor protein
CG51622-OS - Homo


~sapiens
~~~


NOV 13d 375 376 Von Ebner minor protein
~ CG51622-Ol - Homo


~sapiens


NOV 13e ' 377 378 ~ ~ Von Ebner minor protein
275698370 ~ - Homo


_ ", ; ~sapiens
~


NOVl3f 379 380 Von Ebner minor protein
274054187 - Homo


..._...._ ..._.._.....~ ... ..~Sapiens
.._ . ._ _....._.._.....
...... . _.__...
_.. .... .
._ ..._...._
.__.
_......._......
_.._...


.... . .. _ .. ._... .
NO ~CG51622-02 V ~ 382 _ _ .._ _... _
13g 381 Von Ebner minor protein
- Homo


- , Sapiens
_ -


NOV 13h ' CG51622-06383 384 Von Ebner minor protein
- Homo


~sapiens


NOV 13i CG51622-07 385 386 Von Ebner minor protein-
~ Homo


_~ _
-__ _. sapiens
~


NOVl3j CG51622-08 387 388 Von Ebner minor protein
- Homo


_. _.~ ~sapiens
~


NOV l3k CG51622-09 389 390 Von Ebner minor protein
~ , ~ - Homo


_ . ~~. __ _ __ __ _ sapiens
___ _


~ ~_
NOV 131 ' CG51622-10391 392 Von Ebner minor protein~-
' Homo


Sapiens


NOV 13m ' SNP 13375774393 394 Von Ebner minor protein
- Homo


__.
~ _ ~ ~ sapiens
_ __ .. y _ _._
_
4~


_
NOV 13n 3 SNP13375775395 396 _ _
' Von~Ebner minor protein
- Homo


_... ........................~......... ..... ...... Sapiens
_...... _............_._.........._.._._..._......
.._ ....... . _
_..........._.. ~


,~ . ......_.... ...._ _..._._......_.._...
NOV 14 CG51821-Ol a , _ __
' 397 398 Sialoadhesin precursor
j (Sialic acid


binding Ig-like lectin-1)
(Siglec- 1)


(CD169 antigen) - Homo
sapiens


NOV 14b 229823091 399 400 Sialoadhesin precursor
' (Sialic acid


binding Ig-like lectin-1)
(Siglec- 1)


_ _ (CD169 antigen) - Homo
_ _ Sapiens
_


NOV 14c 229823107 401 402 Sialoadhesin precursor
~ (Sialic acid


binding Ig-like lectin-1)
(Siglec- 1)


(CD169 antigen) - Homo
sapiens


NOV 14d 229823095 403 04 Sialoadhesin precursor
~ 4 (Sialic acid


binding Ig-like lectin-1)
(Siglec- 1)


_ (CD169 antigen) - Homo
~~ ~ Sapiens


NO V 05 4 06 Sialoadhesin precursor
l4e CG51821-02 (Sialic acid
4


binding Ig-like lectin-1)
(Siglec- 1)


( CD169 antigen) - Homo
Sapiens


NOVl4f CG51821-03 07 4 08 Sialoadhesin precursor
4 (Sialic acid


b inding Ig-like lectin-1)
(Siglec- 1)


_ ( CD169 antigen) - Homo
y _.__. sa ien
s
P


_
NOV l4g CG51821-04 09 4 10 S _
~ 4 ialoadhesin precursor
(Sialic acid


b inding Ig-like lectin-1)
(Siglec- 1)





CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
~ . ~(CD169 antigen) Homo
sapie
ns _


NOVl4h SNP13382490:411 E412 _
~ Sialoadhesin precursor
(Sialic acid


E
bindingIg-like lectin-1)
(Siglec- 1)


_ ~ ~ (CD169 antigen) - Homo
Sapiens


NOVl4i SNP13382489413 !414 Sialoadhesinprecursor(Sialic
acid


'binding Ig-like lectin-1)
(Siglec- 1)


~ ' ~.- ~ (CD169 antigen) - Homo
~ sapiens


_
NOVl4j SNP13377616415 416 ~Sialoadhesinprecursor(Sialic
acid


(binding Ig-like lectin-1)
(Siglec- 1)


_ (CD_169 antigen) - Homo
~ sapiens


NOVl4k SNP13377617417 418 Sialoadhesin precursor
(Sialic acid


binding Ig-like lectin-1)
(Siglec- 1)


_ (CD169 antigen) - Homo
-~ Sapiens


NO V 419 ~ 420 ' CTCL tumor antigen se57-1
1 Sa CG51992-OS - Homo


Sapiens
_..._.. . . ..... ._.........._..... , .... . .... _......
~... ..._.._._....... . . ....._._..__ . ...... . ... _.....
_............. ....... ..... . . .............. .__...._.
. .. . _........ ._ _ . . _ _ . .... .._
NOV 15b _...... .. ...... .. .
.: ~


~ CG51992-Ol421 ~ 422 CTCL tumor antigen se57-1
- Homo


~ _ , ~~ . _ ~sapiens


_
NOVISc CG51992-02 423 X 424 CTCL tumor antigen se57-1
-Homo
~


apiens


NOV 1 Sd CG51992-03 425 426 CTCL tumor antigen se57-1
~ a - Homo


~ sapiens
__ _. ._. _ _ ~
.v


_ _ _ _
NOVISe CG51992 427 428 CTCL tumoruantigen se57-1~-
04 ' ~y Homo
~


sa iens
_.... _.. _. . _ _ .. p . . _. ..__._.
. __.... ..... ..__
. .. . ..
. ...
_ ...
~


. . . ._ _ ..... .....
~NOVlSf SNP13382486429 430 _ ... . .. _ .. . _._.
. .... .
CTCL tumor antigen se57-1
-Homo


_ _ _ _ ~_ _ sap
Y~ ~ iens


_
NOV 1 Sg SNP 13382487431 432 _ _ _
7 ; CTCL tumor antigen se57-1
- Homo


Sapiens


NOVl6a CG52171-04 433 434 Hematopoietic PBX-interacting


protein - Homo sapiens


NOVl6b CG52171-Ol 435 436 Hematopoietic PBX-interacting
e


protein - Homo sapiens
~ ~


NOV l6c CG52171-02 37 4 38 Hematopoietic PBX-interacting
4


_ protein - Homo Sapiens


NOV 16d CG52171-03 39 4 40 Hematopoietic PBX-interacting
4 s


protein - Homo Sapiens


NOVl6e CG52171-OS 41 4 42 Hematopoietic PBX-interacting
4


~ pro_tein - Homo Sapiens


NOVl6f CG52171-06 43 4 44 Hematopoietic PBX-interacting
4


p rotein - Homo sapiens
_.. . .......... . .__ _. .... .....__...__._....._ ..........
_.._....__...._...___.....___...
. _. ; ... _.... . ..... . ...
~ _. ..._ _. ....
..


._.._. ..__.._..._ _.._.__..
NOVl6g SNP1337462845 4 46 ~ Hematopoietic PBX-interacting
4


_ __ ~ _
_. _ ___ p
~ _ . _ _
rotein _Ho_mo s_a iens
~ ~


NOV l 6h SNP 1337732947 48 Hematopoietic
~ : 4 ~ 4 PBX-interacting


p rotein- Ho
Y~ ~ m
o Sapiens


NOVl6i NP13374627 49 50 _
S 4 4 _
Hematopoietic PBX-interacting


p rotein - Homo sapiens


21



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOVl6j SNP13374626451 452 Hematopoietic~PBX-interacting
~


_ -~ ~protein -_H_omo sapien
" s


NOVl6k SNP13374625453 454 _
i Hematopoietic PBX-interacting


_ _ protein - Homo Sapiens


NOV161 'SNP13374624455 456 Hematopoietic PBX-interacting


_ ~ ' protein - Homo Sapiens
~


NOV 17a ~ CG52534-06457 458 Transfernn receptor protein
2 (TfR2)


_~ ~ - Homo Sapiens '
~ ~


NOVl7b CG52534-Ol 459 ' 460 Transfernn receptor protein
2 (TfR2)


- Homo Sapiens
~ ~ ._ . .


_
NOVl7c . CG52534-02461 462 Transferrin receptor~protein
2 ~(Tfl22)


- Homo Sapiens


_
NOV 17d ~ CG52534-03463 464 Transferrin receptor protein
2 (TfR2)


- Homo sap
~ ie
ns


NOV 17e 3 CG52534-04465 466 _
~ ~ _
Transferrin receptor protein
2 (TfR2)


- ~ i - Homo Sapiens
Y


_
NOV 17f CG52534-OS 467 468 Transferrin receptor protein
2 (TfR2)


_, ", _", ~ - Homo sapiens
, ~


NOVl8a CG52979-03 469 ' 470 G antigen
; family C l protein


' (Prostate-associated gene
protein 4)


(PAGE-4) (PAGE-1) (JM27)


_ _ ~ (GAGE-9) - Homo Sapiens
~ ~ ~ -~ ~~ 4 Y ~
-


NOV 18b CG52979-O1 471 472 G antigen family C 1 protein
: I


i (Prostate-associated gene
protein 4)


(PAGE-4) (PAGE-1) (JM27)


(GAGE-9) - Homo Sapiens


NOV 18c CG52979-02 473 474 G antigen family C 1 protein
'


(Prostate-associated gene
protein 4)


(PAGE-4) (PAGE-1) (JM27)


_ _ _~, _- . -__~ (GAGE_-9)_- Hom_o_sapiens
~~ . , _
~ ~
-


NOV 19a CG52988-02 475 476 _
' ; G antigen
family C 1
protein


(Prostate-associated gene
protein 4)


( PAGE-4) (PAGE-1) (JM27)


_ ( GAGE-9) - Homo Sapiens
. ......~~........- ... ....._..~_... -
_ ......._
..__
..
.


NOVl9b 4 77 47g G
CG52988-03 ~tigen family C 1 proteim.
....... ._... .


( Prostate-associated gene
protein 4)


( PAGE-4) (PAGE-1) (JM27)


_ _ _ ( GAGE-9) - Homo Sapiens
-


NOV 19c CG52988-04 79 480 G antigen family C 1 protein
i 4


( Prostate-associated gene
protein 4)


( PAGE-4) (PAGE-1) (JM27)


_ _ _ ( GAGE-9) - Homo Sapiens
4~ -


NOVl9d CG52988-06 81 4 82 G antigen family C 1 protein
4


( Prostate-associated gene
protein 4)


i( PAGE-4) (PAGE-1) (JM27)


( GAGE-9) - Homo Sapiens


22



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV CG52988-07 483 ~ 484 G antigen Yfamily C l protein
l9e ~


(Prostate-associated gene
protein 4)


(PAGE-4) (PAGE-1) (JM27)


_ , (GAGE-9) - Homo sapiens
~


NOV CG52988-O1 485 486 G antigen family C 1 protein
19f


(Prostate-associated gene
protein 4)


(PAGE-4) (PAGE-1) (JM27)


_ _ , (GAGE-9) - H_ omo Sapiens
~ '
~


NOVl9g CG52988-OS 487 488 G antigen family C 1 protein


(Prostate-associated gene
protein 4)


(PAGE-4) (PAGE-1) (JM27)


_ _ ... .
..............._............._................._..._..................._...
_.... .. . ! (GAGE-9) - Homo Sapiens
..___.. ~ ..._......
............ ....._......
_ _
~


NOV20a CG53449-04 489 490 ~ Butyrophilin like receptor
' - Homo


_ _ _ y~ sap_iens
~ ~ '~


NOV20b CG53449-03 491 492 ~ Butyrophilin like receptor
- Homo


__ sap_iens_ _ _


NOV20c CG53449-O1 493 494 ~Butyrophilin like receptor
- Homo


'~__.. _ ~ , ~ Sapiens


NOV20d CG53449-02 495 496 ~Butyrophilin like receptor
- Homo


_ ~sapiens
~


NOV20e CG53449-05 497 ~ 498 i Butyrophilin like receptor
' ~ - Homo


' _ Sapiens
___ _ ._ _ . _


NOV20f v CG53449-06 499 500 Butyrophilin like receptor
- Homo


~sapie
~ ~ ns _ __


NOV20g ~ SNP13382434501 ~ _
~ 502 iButyrophilin like receptor
- Homo


_ sapiens
~ ~n
'


NOV20h SNP13382441 503 504 Butyrophilin like receptor-Homo


_.........._..__..._........................................._..............._.
............... ......... Isapiens. ........
_.......... .....~. ......
...................................._....
_ ._._ _ ._..._..._........
_....


NOV21 CG53908-O1 SOS j 506 Netrin receptor Unc5h1 -
a Mus


~ _._ _ _ musculus
~_ _ .~ _ ._ ~_.
~ Y _


_ _ _ __
NOV2lb _ 507 508 __
~ __
~ CG53908-02 'Netrin receptor UncShI
- Muse


_ musculus
~~ ~


NOV2lc ' 306075989 509 510 ~Netrin receptor UncShl
- Mus


~musculu_s _
~- ~
J


NOV2ld ~ CG53908-03 511 512 INetrin receptor
~ UncShl
- Mus


musculus
_ _.. _ ., ... .............._.._.. .. ....._...., _ .... . ..
. _. ...... _......... ._ ~ . .. . ..._. ..........__
_ . . _ ..__...._........... ..... .. . .. ....... _.._ ...
. .. .. ...... . _. ... ....._. .....
.... _ _.... . . _ .
_ .


NOV21 CG53908-04 513 514 .
a Netrin receptor Unc5h1 -
Mus


musculus


NOV2lf CG53908-OS SIS 516 NetrinreceptorUnc5h1 -Mus


~musculus


NOV21 ' CG53908-06 517 ' 518 Netrin receptor UncShl -
g Mus


~musculus


NOV2lh CG53908-07 519 ' 520 Netrin receptor UncShl -
Mus


_. . _.__ _.. . ......... . . ~~musculuS.... ..... .
............. ........ _ _.......~._... _...._.. .. . __...
. _............. ...................._. . .... ......... _ ... _...._
......_ . _... ...
... . ...
. .


NOV2li SNP13382444 521 '522 NetrinreceptorlJncShl -Mus


musculus


23



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV2lj SNP13375491~523~ ~ 524 ~Netrin receptor UncShl
- Mus


_ ___ ~ musculus


NOV2lk SNP13375492525 526 ~Netrin receptor UncShl
- Mus


_ ~muscul
us


NOV211 ; SNP13375493527 528 _
Netrin receptor Unc5h1
- Mus


musculus


_
NOV2lm SNP13375135529 530 ~Netrin receptor Unc5h1
- Mus


_ musculus


NOV2ln SNP13375136531 532 Netrin receptor UncShl
- Mus


~musculus
_.. .. .. _._._......_ _.... . . ... .... ... ._ .. ..
. . ....._ ._. __......._..._.... . ...
. ........._...... _. . _
_ ........ .. . _ . ..........
._.. _._....


.
NOV2lo SNP13375137~533 X534 ............ . _ _.__.
_~~"......_._.._.___.
___. _ ._....... .. ..
... ._
Netrin receptor UncShl
- Mus
~


-- musculus
_ y


NOV2lp ~ SNP13375138535 ~Netrin receptor UncShl
536 - Mus


_ musculus
w


_ __
NOV2lq ~ SNP13375494~ 537 Netrin receptor Unc5h1
~ 538 - Mus ~


musculus


NOV2lr ' SNP13375495539 540 Netrin receptor UncShl
- Mus


. .. ..... _
~ _ ........ musculus
... ~


NOV2ls SNP13375140541 ~ 542 Netrin
receptor UncShl - Mus


_ _ _ __~ ~ ~v ~musculus
S


~ _
NOV2lt SNP13375139543 544 m ~ Netrin receptor Unc5h1
- Mus~__


~ musculus


NOV2lu SNP13375496545 546 Netrin receptor Unc5h1
- Mus


_ - T~ musculus
Y


NOV22a CG53944-02 547 548 human secreted polypeptide
' - Homo


~ sapiens
_.. ._.. .......................,~._ .. _... ........... .. ... .
......_...............
_.._. .... . .. .......
.... .. ...~_............ .. . ....._.....
_ _..._... ..
.;


...._ ......_ ......_....
NOV22b CG53944 549 ~ 550 ~ ._..... _ ............._..........
O1 .... _... ..
human secreted polypeptide
- Homo


-_ ___~____~ _ __ _. _sapiens


NOV23a CGS_4308-04551 552 Serpin B12 - Homo sapiens


NOV23b CG54308-O1 553 55_4_ S_er_pin_
B12 -
Homo sapiens


_
NOV_23c,_~G54_308_-02_55_5 556 _
_
S
erpin B
12
-
Hom
o
sap
ie
ns


~!NOV23d CG54308-03 _ _
557 558 ~ _
_
_
__
_
_
_
Ser
pin B12 -Homo sapiens
~~


NOV23e CG54308-05 559 560 _
Serpin B12 - Homo sapiens


NOV24a CG54764-02 561 562 Calgizzarin (Endothelial
monocyte-


activating polypeptide)
(EMAP) -


__ _ a Mus
~ musculus


NOV24b CG54764-O1 563 564 _
Calgizzarin (Endothelial
monocyte-


activating polypeptide)
(EMAP) -


_ _ Mus musculus


NOV25a CG55033-04 565 566 Human toll like receptor
like


~ molecul
e 5
(T
LR-L5)


NOV25b CG55033-05 567 568 _
_
_
Human toll like receptor
like


_ ' ~ molecule 5 (TLR-L5)
~ J


I~TOV25c CG55033-Ol 569 570 Human toll like receptor
like


molecule 5 (TLR-L5)


24



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV25d 237376826 571 ~y~ ~u 572 Human toll like receptor
like


_ _ _~ _mole_cule 5 TLR-L_5 _
( )


NOV25e 310658403 573 574 Human toll like receptor
like


__ _ _ ' J molecule 5 (TLR-_L5)
~


_
NOV25f 317980876 575 576 Human toll like receptor
~ like
1


molecule 5 (TLR-L5_) _


___
NOV25g 318018151 577 ~ 578 Human toll like receptor
like


molecule 5 (TLR-L5)
__ _ _ _ . _ _ _ __ . . _.. _..... ~._..._. ~.
_ ._ _. ~ _.


._
NOV25h 318176316 579 580 ~.._...__ _..___....._
Human toll like receptor
like


molecule 5 (TLR-L5)
_.... ..__.... ....._. . _. __ _. . _ . _ . __........_.
_... . _ . _._.. _ _ . .. ._.
_._...._. _ _. _.. . ....


i CG55033 H
NOV25i -02 581 582 ~ p
. .. . _ ....._ ........
_ ..
uman toll like rece for
like


- ,- molecule 5 (TLR-L5)
X


NOV25j CG55033-03 583 Human toll like receptor
584 like


molecule 5 (TLR-L5) _


NOV25k CG55033-06 585 586 Human toll like receptor
r like


molecule 5 TLR-L5
_ _ _ _. ___ _.._._ __....._.__..__~ __._._..__ )__-_-.-_.___.______...


NOV251 CG55033-07 587 588 Human toll like receptor
like


_.__._.
._. molecule 5 (TLR-L5)


NOV25m CG55033-08 589 590 ! Human toll like receptor
like


_ _ r_~_._ 9 molecule 5 _TL_R-L5 _
m~ _ ___ _~ ~ _ _
_._ ( )


_ _ _
NOV25n CG55033-09 S91 ~ 592 Human toll like receptor
' like


_ ~ molec_ule _5 (TLR_-L_5)
_
a
-


NOV25o CG55033-10 593 ! 594 __..__._.~..~_
! Human toll
like receptor like


_ _ ~ ~ molecule 5 (TLR-L_5) _


NOV25p CG55033-l 595 596 Human toll like receptor
1 like


_......__~..
..._......_......_....._....._...._...... . ..._..................molecule 5
TLR-L5
... ... _. .... __.... _........._......._....(
.._._.._... ............._ __ ..)..
_..


.... _._...._..............._.............
NOV25q CG55033-12 597 598 Human toll like receptor
~ like


_ molecule 5 (TLR-L5)_


NOV25r CG55033-13 599 600 Human toll like receptor
~ like


molecule 5 (TLR-L5)


NOV25s CG55033-14 601 602 Human toll like receptor
like



. molecule 5 (TLR-L5)
r


NOV26a CG55117-04 603 604 Prominin-like protein
a 1 precursor


(Antigen AC133) (CD133
antigen) -


_ Homo Sapiens


NOV26b CG55117-O1 605 606 Prominin-like protein
~ 1 precursor


(Antigen AC133) (CD133
antigen) -


'
~ Homo sapiens
'


NOV26c CG55117-02 607 Prominin-like protein
608 1 precursor


(Antigen AC133) (CD133
antigen) -


_ ___ _ Homo sapiens
' ~


_
NOV26d CG55117-03 609 Prominin-like protein
~ 610 1 precursor


(Antigen AC133) (CD133
antigen) -


Homo sapiens
~ 4-


NOV26e CG55117-05 611 612 Prominin-like protein
1 precursor





CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
(Antigen AC133) (CD133
antigen) -


_ _ ' Homo Sapiens
_ __._. __._ a ~ - _


NOV26f SNP13382440613 614 Prominin-like protein
1 precursor


(Antigen AC133) (CD133
antigen) -


Homo Sapiens
,


NOV26g SNP13382439615 616 Prominin-like protein
1 precursor


(Antigenp 0133) (CD133
antigen) -


Homo sa iens
_.. .._ .._.. __......._..._.. _ _ _...... . . . _._......
._ _ .... ... ._ . . .. ... _ .. . ...._...
...._. . . ... ..... .. ... . _... .. ..._ . . _.._
.. . _.- _.. ..__.._. . .... ..... .. . ... _ .__
_ . ... . . . .
_._._.
..


NOV27a ~ 617 8 PLVAP protein Homo Sapiens
CG55193 61
04


- ~ (Human), 455 as _


NOV27b 214575880 619 620 PLVAP protein - Homo
sapiens


_ ~ __ (Human), 4_55 aa__ _
-~~ _


NOV27c 214458684 621 622 PLVAP protein - Homo
sapiens


_ (Human), 455 as
-


NOV27d 214458688 623 624 PLVAP protein - Homo
Sapiens


_ ... .. . ........_........ ............ ~Human),..455....aa.._..
..._.. .. ..............._.._...._._. _...... __....._....
.. ..... __....~ .~ ... . . ...._.
..._...


NOV27e CG55193-Ol 625 ~ 626 PLVAP protein - Homo
sapiens


_ (Human), 455 as __ _
~


NOV27f CG55193-02 627 628 PLVAP protein - Homo
Sapiens


_ ___ ~ , ~ (Hu_man), 455 as _____
~~


NOV27g CG55193-03 629 630 PLVAP protein - Homo
~ ~ Sapiens


_ ~ .~ (Human), 455 as
Y~ ~ ~ Y


NOV27h CG55193-05 631 632 PLVAP protein -Homo Sapiens


_...... ... .. ._. __. ~ _ _. (Human), 455 as
... .. ...... _._
.......
_._. _.


NOV27i CG55193-06 633 634 PLVAP protein -Homo sapienS
~


_ _ ~ (Human), 455 as
~~~~


NOV27j CG55193-07 635 636 PLVAP protein - Homo
Sapiens


(Human), 455 as


NOV28a CG55256-07 637 638 Apolipoprotein E receptor
2


precursor - Homo sapiens


NOV28b CG55256-10 639 640 Apolipoprotein E receptor
2


_ ............_..........._..................._.._..___... _
precursor..- Homo Sapiens
.. ......_......._ _ ,.. ._........ .. _.
._.. ......
. ....
..._.


NOV28c 272511714 641 X 642 Apolipoprotein E receptor
" ~ 2


precursor - Homo Sapiens
._ _ _ _ _ _. a __ ___ ~ _ ~ __ _


NOV28d CG55256-Ol 643 644 Apolipoprotein E receptor
2


precursor - Homo sapiens
_


NOV28e CG55256-02 645 646 Apolipoprotein E receptor
~ 2


- ~ _ _ _ precursor - Homo Sapiens


NOV28f CG55256-03 647 648 Apolipoprotein E receptor
~ 2


_.. ._ _ _ _.. _ .. _ ...l lprecursor -_ Homo. sapiens
_ ..._ .. _ ... _ ._. ..
.. .__. _..
..


NOV28g CG55256-04 649 650 Apolipoprotein E receptor
2


_ ~ _ prec_urs_or_- Homo sapiens
_
~


NOV28h CG55256-05 :621 652 Apolipoprotein E receptor
2


~ precursor - Homo Sapiens


NOV28i CG55256-06 653 ~ 654 Apolipoprotein E receptor
2


26



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
. .__........._~.._.. _..~~ _ precursor- Homo sa .iens
...__...__......... ' ,." _...___...,..
__. ..... _,.~1
_..._"~._...__......._..___..._......._.__....,........._._....._....~
.... _ .. - ~ .
~


~,_
NOV28j CG55256-08 655 656 Apolipoprotein E receptor
2
_ . _..... -. . _.._ ~
.__... ~.... - . .___ .. precursor - Homo Sapiens
_.__ _..___._....... . _..
_ ._ _ . ___ .
. .


CG55256-09 657 p
NOV28k 658 .
~ CG55256-11 __ ~, _ . ..._ .. __.._ ..._.._..._
NOV281 X 659 660 _._.__ .... . . ..
~ Apoli oprotem E receptor
_ SNP13382497 661 662 2
NOV28m precursor - H_omo_s_apiens_
~ SNP13382498663 664 Apolipoprotein E receptor
NOV28n v _~ 2~
~ _ .. . .. _precursor - Homo Sapiens
. _ . _ ,.._.. ..._.. Apolipoprotein E receptor
_ . _..... .. . ..... 2
. . _... ....._ .. . . ._.....precursor - Homo Sapiens
_..,.. . _. . .. . .. . ... _
_.. . _ Apolipoprotein E receptor
... .. 2 ~~
_.._ ~ precursor - Homo Sapiens
... _.......... .. .. ..._......
. ._ .._._ .. .. _


. ._...._........ . ..
NOV29a CG55776-O1 665 666 _.... ... . .....
_. . ... ...:.... _. _ ..... Human osteoclast protein
..__ ....... . . ...~ _....(OCP)
.... . . . ... .. _ . .. _..._... .. ...
. _.._........._.__........ ...,.. _.. ..... ......
... ......... .._... _...,....
.._ ..
..
.


..
NOV29b 2482.1.01,67,.,....,.,-667,.668 .
. ......... _ ... _......
_ ..._..... ; w._........ ..._ . _......._. .....
. .._. ....... ................. _ ..
. _n _
Human osteoclast protein
(OCP)
........... .. . ..,...
... . _... . .... . ..
.


, ..._.. ..........,. ...
NOV29c 247679561 669 670 ......
.......... :.. ._ ....._.. ~ ..._ Human osteoclast protein
.._.............. _......._..... . ~~ (OCP)
. .~ .... . ..._. . _ _.


NOV29d '248057904 671 672 a Human osteoclast protein
_ ................. .... ... _ ~.. ....(OCP)
... ..... . ........_.... .. ..... . .. ._
. ....... ........ ..............
........ ..... .....
_.. .


.. . ..
NOV29e _248057927 673 . , .,..-674... ..... .. ........
, ,-... . .. .. Human osteoclast protein
. . (OCP)


NOV29f 249239821 675 676 Human osteoclast protein
_.. .... _ ... ... _...... (OCP)
.... .. ._ .._. .. . .. .. __.... _.,
. . . ......".. ......,.........


g ,2480579 .. .. ..,_._..... ........._
NOV29...._......._....._...,~........._..677 ........ Human, osteoclast
protein
..... . . .. ......678 (OCP)q
...2~.._.. . .,......
...._...... ... . ...
....._I


NOV29h 247679541 679 680 9 Human osteoclast protein
.... _. ... ... ~... . (OCP)
. _.. .._... . ..._ . . . _. _ _ ,.. , . , ...
. ..~ . , ... _.. _ . _ . .....
. ~


NOV29i 249116954 681 682 Human osteoclast protein
.......... ;_.... .. .. .._. ......(OCP)
....._......._._.......................__............... . _.... ..
......_.........
. ..........._ _ .. .... "... . .......... ..
.. ...... ...,..... .._... . .............
..u ...


NOV29j ;248210155 683 684 Human osteoclast protein
_. ......___............_................_ .. ._........._...(OCP)
_...... ..._.............._ ................. ........
,....._..............................
..........,........._............_.._ _.... ,_........
_........ .
.
~


, , ..
NOV29k , 685 686-, .,.... ......_.,__ .......
_..._....; 248213764 . j ............_. ..__........
; ........ ...... Human osteoclast protein
.... .. __._._._......... _.. _ (OCP)
............... _ ...... _.. ... . . _.....
.._..............,.._._, .
..


.
NOV291 .248213768 687 .. .,.. .. .. . .._ ....... ..,~,_,..
. .. . , ,... ,688 .- _......_ ...
,-,.,. Human osteoclast protein
(OCP)


NOV29m 248213772 689 690 Human osteoclast protein
~ _ _ a _ .._..(OCP)
__. . .. . .. ....... ...........
_~ .. ......_.,_.._..._........~_.....
- .


.
NOV29n ~248586774~ 691, 692 .. ._. .. . . ........
_............_....._.......... r,......._..... ... ....., ... _ ....
..... .:".~~~...._...._ .... .... .._._.
Human osteoclast protein
(OCP
-........_..._........_._
.
.
..._.
...
.
~


.
NOV29o ?248586793 693 694 ,
_.. .......;............................._ .. .. _
.._..............._ ...
. _ ....
_._ .._.... ......_._ ...._
.. _...,...
Human osteoclast protein
(OCP)
_. ............ .........._..........
_.._....... ......_.
. _........... .


NOV29p 248586820 695 696 Human osteoclast protein
y- (OCP)
-


NOV29q 248586824 697 698 Human osteoclast
_.... . ... rotein OCP
_ _ .. p ( )
_ _ _ ... ...... ....


NOV29r 247679817 699 _ .... _... _..
700 Human osteoclast protein
y ~ ~ (OCP)
~ - ~u


NOV29s ' 248210264. 701 . 702 Human osteoclast protein
, ,. (OCP) y


NOV29t 248210551 703 704 Human osteoclast protein
.- -.. . . . ._.. _._. _._ (OCP)
. . .... _ _ ....._.... ,.........._
y _.. . . __ . ._.._. _.
.


~ 2482 .._. ...._ ....,...
NOV2 ~ .. - -...1.0824705 706 , .. Human osteoclast protein
.... ~~~ _.,_. (OCP)
9u
W ._.


NO_V29v248679541 707_ 708 Human osteoclast protein
X...... (OCP)
~ _ E~_ ... ...._.,__...._....,......
_......._._....._"~,
_,~,. ..~,_-"_.._._ ......
._
_...


NOV29w 709 710
_. .. ' _ ... .__.p ( )
........;'247679454 ...... Human osteocla_st rotein_
.. .x:,_..._...._,. . ._ ......OCP _
...,_,~. . L . .. ._ ...... ..........._._._.._..
__.__.._... _. .


NOV29x 314361407 711 712 Human osteoclast_p
Y Y ~ _ ~ rotein (OCP)
~ ~ ~


_
NOV29y 317803448 713 714 _
... . _~......._ ..__....~ Human osteoclast
_ .._..._. _.._. _.... _____. rotein OCP
........._...__ _. ._.~ ~_. . . . ....._.. _ _ ._ ._
_ . _._.. _. _. . .. . .. p ...
.~ .. _..~.. _ _ )__ . .
...


NOV29z 'CG55776-02 715 "71.6._ Human osteoclast protein
_ ,. ., (OCP)
~- ,
~


N 29aa CG55776 03 717 j 7I 8. Human osteoclast protein
, j (OCP)
-


NOV29abCG55776 04 719 720 Human osteoclast protein
_~ _~ (OCP)
.


NOV29ac~CG55776 OS 721 722 Human oste_oclast protein
~ ~ (OCP) _


NOV29a d CG55776 723 724 ' Human osteoclast protein
06 ' _.. (OCP)


__
NOV29aeCG55776-07 725 ~ 726 Human osteoclast protein
(OCP) -


27



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV29af CG55776-08 727 ~ ~~ Human osteoclast protein
_.. _..........._..' ~ 728 (OCP)
__......_..._.... :.... __ .....
. ._.... ........~,..__.... .. .... . _ _......_
_._..._..._.............. ~".._-
...._,~,_......._.._........._......_.._....
z- ~__~ . .. .. _.....................
_


NOV29ag.".._. CG55776-09..._,729 .. . ,.,. ,._.Human.osteoclast
,.. "_...... 730 protein (OCP)
....__. ~
_ ~
y


NOV29ah 'SNP13376522731 _ ,.732 -~Humanosteo
~ _....__.._: _. c
~. _._._ last
_.._.__ protei
, n
(OCP
)


N_ OV29ai~,~ 733 734 _
SNP13377624 _
~ _
_
Human osteoclast protein
(OCP)'
~


NOV29aj SNP13377625 735 736 'Human osteoclast protein
(OCP)


NOV29ak ~ SN_P1_3376523737 738 Human osteoclast protein
, _- - (OCP)
v


NOV29a1 SNP 7 740 Human osteo
1337 39 clast
6524 pr
otein (OCP)


_ _ "~"~, _
NOV29_am_ _ ~ 742 _
' SNP 1.3376525_ _ __. _
~ ~ Human osteoclast protein
~ 741 (OCP)
~ __


N_ OV29an~ SNP 133765743 744 Human oste_ocl
~ 26 ~~ ast protein (OCP)


NOV2 _ 745 746 _
9ao NP13377626 Human osteoc
S la
st proteinV(OCP)
~


_ _ 747 _ _
_ ~' 748 _
NOV29ap SNP13377641 ___
' Human osteoclast protein~~(OCP)
~


, _
__ . ~ _ _ _ 749 750 _ _.. ..r ...._ _._... __~
NOV29aq SNP13377640 _. ..
._. ... ___~__ _ ~. .~.,n Human osteoclast protein
__ _..~ _ - (OCP)
s.. _ ........ ...._.___..__
_.


NOV30a CG55784-03 751 752 NPEH2 - Homo sapiens
~~ ~


NOV_30b CGS_5784-O1 753 754 NPEH2 - Homo
Y~ 4 ~ sapiens
Y


NOV30c 312000 755 756 NPEH2 -
579 Homo sapiens


_ _ 757 758 ~NPEH2 - Homo sapien
NOV30d ~:3 s
l 1
33
3341 ~ -


30e _ 759 7604-~ _
NO _ ~ -~NP
V _ EH2 - Homo~sapiens
~ 3113
33338


_ _ 761 ' 762 _
_ Y~ 3113 ~NPEH2 - Homo Sapiens y~
NOV30f 46
4 885


N_OV30g~~_ 763 i764~ ~NPEH_2 - Homo sapiens
~~~ _ ~ W
~ 311333299 ~
~
-


NOV30h CG55784-02 765 766 NPEH2 -Homo
_ W.-.__; . __. sapiens
_ _..____.___~_._ - _ ___ _ __ .. .
_. ... . ..
., ____
_
.:


~ ~.__._. __.
NOV30i . 767 768 _NPEH2 - Homo sapiens_ _
SNP13376439
'
-
~


NOV3la CG55790-02 769 '770 ICOS ligand precursor (B7
homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1) (B7RP-1)
-


Homo sapiens


NOV3lb 258668431 771 '772 ICOS ligand precursor (B7
homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1) (B7RP-1)
-


_ Homo Sapiens
_ _ ___ _____. ~ _ _


NOV3lc 309303509 773 774 ICOS ligand precursor (B7
homolog


2) (B7-H2) (B7-like protein
6150)


otein-1) (B7RP-1) -
l


~ _ sapiens
~ ~Homo


NOV3ld 315925314 775 776 ~ICOS ligand precursor (B'7
homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1) (B7RP-1)
-


Homo sapiens _


NOV3le 315970230 777 778 ICOS ligand precursor (B7
homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1) (B7RP-1)
-


_ _ Homo Sapiens


NOV3lf CG55790-O1 779 780 ICOS Iigand precursor (B7
homolog


2) (B7-H2) (B7-like protein
6150)
~


(B7-related protein-1) (B7RP-1)
-


28



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
~.._....~__.~"~ H p.
..... ... ..._.......
. .._ .....~.._.
.. sa lens
_.._. . . .........
.~.._.. y ..._.
mo
~
.


NOV3 CG55790-03 1 .
g 3 781 782 ,.
,.. _.....~_~..._.
_
ICOS ligand precursor
(B7 homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1)
(B7RP-1) -


_ Homo sapiens


NOV3lh CG55790-04 783 784 ICOS ligand precursor
(B7 homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1)
(B7RP-1) -


Homo Sapiens


_
NOV3li CG55790-05 785 786 -ICOS ligand precursor
(B7 homolog
i


2) (B7-H2) (B7-like protein
6150)
~(B7-related protein-1)
(B7RP-1) -


Homo sapiens


NOV3lj CG55790-06 787 788 ICOS ligand precursor
(B7 homolog


2) (B7-H2) (B7-like protein
GI50)


(B7-related protein-1)
(B7RP-1) -


H
omo sapiens
~


NOV3lk CG55790-07 789 790 _
ICOS ligand precursor
(B7 homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1)
(B7RP-1) -


_ Homo Sapiens
'


NOV311 SNP13374852791 792 ICOS ligand precursor
' : (B7 homolog


2) (B7-H2) (B7-like protein
6150)


(B7-related protein-1)
(B7RP-1) -


Homo sapiens


_
NOV32a CG55906-04 7994 Human breast tumour-associated


protein 37


_
NOV32b CG55906-05 795 796 Human breast tumour-as~
~ sociated


% protein 37


_
NOV32c CG55906-01 797 798 V Human breast tumour-associated
;


protein 37


NOV32d 277901794 799 800 Human breast tumour-associated


_ ~ protein
37


NOV32 CG55906-02 e _
801 802 . Human breast tumour-associated


protein 37


NOV32f 230272941 803 804 Human breast tumour-associated


protein 37


NOV32g CG55906-03 805 806 Human breast tumour-associated


._. . _.. ... . .... _.._protein 3 7
. . _ _.._


NOV32h NP13382505 07 ' 808 Human breast tumour-associated
S 8


protein 37


NOV32i NP13382504 09 810 ' Human breast tumour-associated
S 8


protein 37


NOV32j NP13376440 11 812 Human breast tumour-associated
S . 8


protein 37
n
~


.
NOV33a G55908-01 13 ntegrin alpha-7 chain
C 8 814 i precursor-


29



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
. ...~_..___.._._...human .._...........
~ J _... ....... __....,-....._...


NOV33b 253116407 815 816 ntegrin alpha-7 chain
; i precursor-


_ .............._._....._._......___ _ . .. __ _._.. human
.. . .. __ ... _.
.. .._ _._.._
_...._.. _. _..
_ .


NOV33c 253116412 817 818 ntegrin alpha-7 chainprecursor-
~i


~ human _ _
~ _


NOV34a CG56077-06 819 820 _
~ LGI4 (Leucine-rich glioma


inactivated protein 4)
(LGIl-like


protein 3) - Homo Sapiens
~


NOV34b CG56077-O1 821 822 LGI4 (Leucine-rich glioma
~


inactivated protein 4)
(LGIl-like


__ protein 3) - Homo Sapiens
~ -


NOV34c CG56077-02 823 824 LGI4 (Leucine-rich glioma


inactivated protein 4)
(LGIl-like


protein 3) - Homo sapiens


NOV34d CG56077-03 825 826 LGI4 (Leucine-rich glioma


inactivated protein 4)
(LGIl-like


__ _ protein 3) - Homo sapiens


NOV34e CG56077-04 827 828 LGI4 (Leucine-rich glioma


inactivated protein 4)
(LGI1-like


-__ ~~ - 4 protein 3) - Homo Sapiens
. V


NOV34f CG56077-OS 829 830 LGI4 (Leucine-rich glioma


i inactivated protein 4)
(LGIl-like


T.
_ _ protein 3) - Homo Sapiens
_ ~ ~ __- . _ _ . _ _ _


NOV34g SNP13374715831 832 LGI4 (Leucine-rich glioma
~ : ~


inactivated protein 4)
(LGIl-like


_ ~ protein 3) - Homo Sapiens
~ m~ -


NOV34h SNP13374714833 834 LGI4 (Leucine-rich glioma


inactivated protein 4)
(LGIl-like


_ _ protein 3) - _Homo Sapiens


NOV35a CG56110-03 835 836 B7-Hl (PD-1-ligand precursor)
-


~ Homo Sapiens


NOV35b CG56110-07 837 838 B7-Hl (PD-1-ligand precursor)
-


_. Homo Sapiens
-


NOV35c 274082305 839 840 B7-H1 (PD-1-ligand precursor)
-


_....... ... . .. . ,",... Homo sapiens~_
_,~,_>,.....__..._......_.._._......._....._._..
_... ....... . ._
... .. ......__..


NOV35d CG56110-O1 841 842 B7-Hl (PD-1-ligand precursor)
' -


_ _ _ _ _ H_omo_sapiens _
'


NOV35e CG56110-02 843 844 B7-H1 (PD-1-ligand precursor)
-


Homo Sapiens


NOV35f CG56110-04 845 846 B7-Hl (PD-1-ligand precursor)
~ -


Homo Sapiens


NOV35g CG56110-OS 847 848 B7-H1 (PD-1-ligand precursor)
~ -


_. _.._..._..._ .__ _._ _...~. _ Homo sapiens __... _
_. . _ .... _ ._.__ . .._ ... _. _.._ . _.. .
_. _ _ ..._ .__ _..._ . _...._


NOV35h CG56110-06 849 850 B7-H1 (PD-1-ligand precursor)
-


_ _ ._ . ~- ~. _~ Homo sapien_s _
, -
_


NOV36a CG56383-02 ~ 851 852 Prickle-like 1 (Drosophila)
- Mus





CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
~.,~": ... _.... .. musculus-(Mouse),.,879.
__............._ i aa, _.. _......~~ .
..._.... _ _ ... ..
~,.~....~.


NOV36b CG56383-O1 853 ~ 854 ( Prickle-like 1 (Drosophila)
' - Mus


_ ...__ ~ musculus (Mouse) 879
.. _ ...._................._....__.._..._....._as _
......_....-.............._ .._._... __.........._.........
_._.... ....
.. ...


NOV36c SNP13382513855 856 Prickle-like 1 (Drosophila)
v - Mus


m_usculu_s (Mouse)_,
~ -- 8_79 _aa __ _


NOV37a CG56449-04 857 858 MEGF6 - Homo sapiens
(Human),


1246 as
'


NOV37b CG56449-09 860 MEGF6 - Homo Sapiens
~ ~ 859 (Human),


~ 1246' as


_ _
NOV37c 191887507 861 ~ 862 MEGF6 - Homo Sapiens
' (Human),


_ _ . ._ ...........~ . ... .... 1246, as _.. .... .._
. . _ .. . ...._.... .. _ . .. _... .........
.._. .. ... .. .._ . _ .


NOV37d 316351371 863 ~ 864 MEGF6 - Homo Sapiens
~ (Human),


_ ___..__ ___ 1246 as ____~ _ . _ .
__ _


...___.._ .__. ___ 865 866 MEGF6 - Homo Sapiens
_.._.~ 316935396 (Human),
__
NOV37e


1246 as


NOV37f 317004318 867 868 MEGF6 - Homo Sapiens
(Human),


_ _._ _ _ _. . _ _. 1246 as .., .. .. ~..
___. ~ _ _ ~ . __ _


_ .._ __. _ 869 870 ' MEGF6 - Homo sapiens
_. CG56449-Ol (Human),
NOV37g


1246 as
- .._..... _ ._ . _.. ... __......_.
_.. ..... .
......


NOV37h CG56449-02 871 872 MEGF6 - Homo Sapiens
(Human),


1246 as
_ _ _ ~~__ _ _.. _ ._. ._ .
.


_.__._ _ _ _._ ~_ .
....~ .__~__ . __.____ MEGF6 - Homo sapiens
. CG56449-03 873 874 (Human),
NOV37i
~


1246 as


NOV37j CG56449-OS 875 876 MEGF6 - Homo sapiens
(Human),


1246 as


NOV37k ___.._ . 877 878 MEGF6 - Homo Sapiens
~~ (Human),
CG56449-06


_..._.........................._......................... __...._.1246 as
- _.__ . ._.. 4
-


NOV371 CG56449-07 879 880 Homo Sapiens (Human),
' MEGF6 -


1246 as


NOV37m _ 881 882 MEGF6 - Homo Sapiens
' CG56449-08 (Human),


1246 as


NOV37n CG56449-10 883 884 MEGF6 - Homo sapiens
(Human),


_ 1246 as
--


NOV37o CG56449-11 885 886 MEGF6 -Homo Sapiens (Human),


_. .. .. ..._...._. ...... . 1246, as . _... .....
... _... _ ... . _ _..._.. _ .. _. _.. .. ..
....... . .
..._. _....


NOV37p ~ SNP13382514887 888 MEGF6 - Homo Sapiens
(Human),


~ 1246 as


NOV38a _ ~ 889 890 Claudin-19 - Homo Sapiens
_ CG56594-O1


NOV38b 277685552 891 892 Claudin-19 - Homo sapiens
, _


V38c 277685649 893 Claudin-19 - Homo Sapiens
NO 894 ~-


_ 277685616 895 896 ~ Claudin-19 - Homo sapiens
NOV38d


NOV39a CG56653-08 897 ~ 89_8 ficolin-l precursor
._. _____._____~___~.__~ - _______~~.
___.___._ . -. N ~~_~_.___.~_...______


~ ~21 900 =
NOV39b 4_374274 899 ,~ _ ,
_ ~~ ficolin_1 r_e_cur_sor
~z
-
~
p
--


NOV39c 214374256 ' 901 902 -
__
ficolin-I precursor


31



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV39d 214374252 9_03 ~ 904Y ~ ficolin-1 precursor _
_~ ... , _._......_._..._....._. ~ _ _ _
. .. ......,...._.. _.... ..
~ u ~ ~
u ~~


NOV39e 214458492 905 906 ~ficolin-1
' _......__.__.._._ ,.......precursor
_. . . _._ _.._...,_.__ _..._......_ _ ,__.......
... _.
._.___......_.........__..._....._.._.._._,_.
.. . . .._.. _..


NOV_39f 214458488 907 _908 _ ficolin-l precursor
~ ~ 4
~


NOV39g 214374236 90~ 910 ~
a ' ficolin-l precursor
rv


NOV39h CG56653-Ol 911 ~ 912 ~
' ~ - ~ficolin-_1 precursor
~


NOV39i CG56653-02 913 914 ficolin-_1 precursor
a . .~_,___.___"~x
4 ~_ ______
___ _ ,
~
~


NOV39j CGS 915 , ~ficolin-1precursor
665 ..._.____._~ 9146
~ 3-03 .. ,
~ .
. , ~
~


N_OV39k _ -. 918 ficolin-1 precursor
v _ 917 .
_
CG566_53-04
'


NOV391 CG56653-OS 919 920 ficolin-1 precursor
'


_ CG_56653-06921 ~ 922 ficolin-1 precursor
NOV39m ~ ",.~ ...,__...",.,1.,.,._,...__...W..,_-..
~- : 1 ,. t .. ..
~ ~ ~ .__ . ,.
." ...
.._,
.


~yyye """ ""
NOV39n CG56653-07 9_23 i 924 .__
e _ ...____ ' ~ ...
~~ . . ... .~
~~y~ ~y .~....
...
~ficolin-1 precursor
~~


NOV39o CG56653-09 925 '926 ficolin-1 precursor
' ..._ ._ _. _ _ _._
_, ... _ ~._
.. _,.,
_ " .
_~


NOV39p CG56653_-10927 ~928 ~ficolin-1 precursor
_ ~
~


NOV39q C_G56653-11929 ~ ficolin-l
~ 930 precursor
~~ ~ ~


NOV39r CG56653-12 931 932 ficolin-1 precursor
__._____. J ,., __.___ _ ~_ _ _
~ ___ ___ ... ._ . _...,__._~
_..,_. ,
, ~


", ",
NOV40a CG56806-Ol f=.",X934 -
933 _._.._. .
Heparan sulfate 6-O-sulfotransferase
~~


_ ........................_., ........
_.._..... _...._....,:........,_...,......_......'ens
_ 3..- Homo saps
... ...........
.. y
~


NOV40b 248061366 935 X936 ~Heparan sulfate
e 6-O-sulfotransferase


__ _ ~ ~ 3 - Homo_s_apie_ns_ __~~


_
NOV40c 246837961 937 938 ;Heparan sulfate 6-O-sulfotransferase


3 - Homo sapiens
~


NOV40d 246837965 939 940 'Heparan sulfate 6-O-sulfotransferase


~ __ _ _-_ _, _.. ~__.._...__-_..ie
_._,. ___ _ n_ s _ _
. ~.. 3 -_Homo sap


_
NOV40e 246837975 941 942 _
~ ~ _ __ _
Heparan sulfate 6-O-sulfotransferase


_.__. ._. ._... ................__.._.. ._., _._. 3....-..Homo sapiens
.,_, , ...... _, ,_. ,. ...... .._......_ ......
_... _. . ".._.,........ _ ... . _., _


NOV40f 248061376 943 944 Heparan sulfate 6-O-sulfotransferase


_ 3 - Homo sapiens


NOV40g SNP13381685945 946 Heparan sulfate 6-O-sulfotransferase


3 - Homo sapiens


NOV40h SNP13381686947 948 Heparan sulfate 6-O-sulfotransferase
~


_ _ ~3 - Homo Sapiens
~ ~


NOV41 a CG56878-O1 949 950 Protein OS-9 precursor -
' Homo


_... ....... _....._ ._._.....__,._....,_.._..._,...._Isapiens _..........
. ... . .._ .. ,._...._~~....,._..._,_ ........... . _._,._._
_ .. ._,...._ ._...._ ".__..
___ ~


NOV4lb 175070399 951 952 Protein OS-9 precursor -
Homo


_ _ sapiens


NOV4lc 175070432 953 954 Protein OS-9 precursor -
Homo


sapiens


NOV41 d 175070419 955 956 Protein OS-9 precursor -
Homo


_ _ ,. . __ ~,_ s_apien_s _
. 4 ~~


NOV4l a 175070438 957 958 ~ Protein OS-9 precursor
; ' - Homo


__. . _ ... _... _ ..._. Sapiens _...,. .._ . ....
_ .. ...._ _ _.._.._ . _ _____
_.. .. _.__


NOV41 f 175070408 959 960 ~ Protein OS-9 precursor
- Homo


Sapiens


32



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV4l g CG56878-02 961 ~ .,' Protein OS-9 precursor
962 - Homo


_ sapiens


NOV4lh CG56878-03 963 964 Protein OS-9 precursor
- Homo


_ _ __ _ _ _ _sapiens


NOV41 i _ 965 i 966 , Protein OS-9 precursor
CG56878-04 - Homo


. _ _ _ sa
~ p
iens


NOV41 j ~ SNP 13382511967 , 968 _
_
Protein OS-9 precursor
- Homo


_ - ~ _ ,Sapiens
- ~ ~ ~


NOV4lk SNP13382506969 970 Protein OS-9 precursor
- Homo


~sapiens
..._.... ..__ _.. _... _.._ . ......_. ._.... ._.....
. _ . ._ . _.._.....
.. .. . __... ._.
.....


NOV42a ' ,
~CG56904 971 972 . ........_ ..._._ .._.........
O1 _ . . _.. . _ ...._. ......
~ secreted protein LP255(a)


NOV42b 272355087 973 X974 'secreted protein LP255(a)
... ~~


NOV42c 246862506 975 976 ~ secreted
~ ~ ~ pr
otein LP255(a)


_
NOV42d CG5_6904-04977 978 _
. .., _....... .. .~......__..._........_..__._. _ _
. ..._..... ~ secreted rotein LP255
_.. a
~ .. .,........_......__.._..p
.
~
~


",~.~,~._.._....._._._._...
NOV42e 268824694 ' 979 ~ 980 _..
.._. _... .._.. ....... ...._._..._... _~ ....._ ... ...
_. .. _ ... ...............
........._.... secreted protein LP255(a)
. _...........


NOV4 _..... ... .. ............
2f 283146542 9,8.1 982. _.....
.. secreted protein LP255(a)
....


NOV42g 308521214 983 , 984 secreted protein LP255(a)
_
~


_
NOV42h CG56904 985 986 secreted protein LP255(a)
02 a ~ ~
......
~ a


NOV42i CG56904 987 988 secreted protein LP255(a)
03
.


NOV42j CG56904 989 990 secreted protein LP255(a)
05 _ a ..


NOV42k CG56904 991 992 secreted protein LP255(a)
06 ~


NOV421 CG56904 993 994 secreted protein LP255(a)
' 07 _ . _ _ _. .. _. ... .._.. .
~ ~ ........ . ... .


_...... . ._...... ..._
NOV42m CG56904 995 996 ....
~ 08 ~ secreted protein LP255(a)


NOV43a CG56914 997 998 Hemicentin - Homo Sapiens
Ol _ .., _ ..
. A _.
_... ~ ...._..Y
_. A


NOV43b 262802367 999 ~ 1000
...._.__..,......................._.._._._................
_. .._....... ~,-;. _..... Hemicentin -~~
_ . .__..... _..~_, .~ Homo sapiens
...... .... .... ...... .. .
_ ... . ....
.._..


.... ...
NOV43c CG56914-02.....,..,...~1001 1002 . ..._ .. ._.........
' _ ~ Hemicentin - Homo Sapiens


NOV43 d CG56914-031003 1004 Hemicentin - Homo Sapiens
_ ~~


NOV44a CG56959-02 1005 1006 Synaptotagmin 10 - Rattus


__ __ _ ___ norvegicus
~ ~ __~ ~
~


NOV44b CG56959-Ol 1007 1008 Synaptotagmin 10 - Rattus
~ ~


_ norvegicus


NOV45a CG57111-O1 1009 1010 Protocadherin I3 - Homo
' ~ sapiens


~ _ ._. _ _ _ _ ~ (Human),
-~ - 947aa


_
NOV45b 277726328 1 OI 1012 _
' 1 Protocadherin 13 - Homo
~ Sapiens


(Human), 947 as
~ ~


NOV45c CG57I 11-021013 1014 Protocadherin ~l3 - Homo
sapiens Y


(Human), 947 as


NOV45d CG57111-03 1015 1016 Protocadherin 13 - Homo
' sapiens


( Human), 947 as


NOV45e CG57111-04 1017 1018 Protocadherin 13 - Homo
sapiens


~( Human
~ ), 947 as


NOV46a CG57409-05 1019 1020 _
J Glycosyl-phosphatidyl-inositol-


MAM - Homo. sapiens


33



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV46b 277731446 l 1022 ~ Glycosyl phospha
021 tidyl-inositol-
~
.


~ p
MAM - Homo sa iens


NOV46c CG57409-07 1023 1024 ; Glycosyl-phosphatidyl-inositol-


_ __ _- -_ ~MAM - Homo Sapiens
T ~


NOV46d 312102874 1025 1026 Glycosyl-phosphatidyl-inositol-
'


_ ~ MAM - Homo Sapiens
~ ~


NOV46e 312102899 1027 1028 ~
~ Glycosyl-phosphatidyl-inositol-


_ . __ ~._~M~ - Homo Sapiens
.


NOV46f CG57409-O1 1029 1030 Glycosyl-phosphatidyl-inositol-


_... .
............__...._.........._..........._..........._.__........_.........._.
.. ~.' Homo.sapiens _ ......
...._...._.._......__....._.. ... .._._....._.......___
~.
....._..
._


NOV46g CG57409-02 1031 1032 Glycosyl-phosphatidyl-inositol-
~


_ _ _ _~ ~ ~~~_ ~~MAM_-_Homo sapiens
~~ ~


NOV46h CG57409-03 1033 1034 Glycosyl-phosphatidyl-inositol-


_ ~ MA_M_- _Ho_m_o s_ap_ie_ns


NOV46i CG57409-04 1035 1036 ~ Glycosyl-phosphatidyl-inositol-


_ _ , MAM - Homo sapiens


NOV46j CG57409-06 1037 1038 Glycosyl-phosphatidyl-inositol-


_.. _ ...._ . ._.. _... ~M. Homo sapiens . .....
. . . . .... _ ~ .... .. .. .
.. . .. ... ... ._
. .. .... _.
.


NOV46k CG57409-08 1039 1040 Glycosyl-phosphatidyl-inositol-


_. __
_._ _ __. ___ _ _ ~ MAM - Homo Sapiens
___ _ _~.. ______ __ ~-___ ~.. __ _ ___ .._~~._
_ .~ _.. ____~


NOV47a CG57448-O1 1041 ;1042 Human cadherin-1 (CDHN-1)
~ ~


___ _ _ _ protein _


NOV47b 247846705 1043 1044 Human cadherin-1 (CDHN-1)
.


,_ _ protein _~
~ ~


NOV47c 247846708 1045 1046 Human cadherin-1 (CDHN-1)
;


protein


NOV47d 237580295 1047 ' 1048 Human cadherin-1 (CDHN-1)
~ ~


~~ protein


__ ~
_.______


NOV47e 237579512 1049 1050 Human cadherin-1 (CDHN-1)


_ ~ protein


NOV47f CG57448-02 1051 1052 Human cadherin-1 (CDHN-1)


_ _ -~~protein


NOV48a CG57574-03 1053 1054 ~ Tectorin beta -
Homo sapiens


NOV48b _ 1055 ~~_1056_
V CG57574-O1 s Tectorin_beta - Homo sapiens


NOV48_c CG57574-02 1057 1058 I Tectorin beta - Homo sapiens
~


NOV48d CG57574-04 1059 1060 Tectorin beta - Homo Sapiens
~ ~


NO_V_48e CG57574-05 _10611062 Tectorin beta - Homo sapiens
~


NOV49a CG57689-O1 1063 1064 Inferred: hemicentin - Mus
a !._ _..._ A _ !4 musculus
_.. . . _ __ _. b ,
_ _...._ .._ ~


NOV50a CG58567-O1 1_0651066 pHuman cadherin (CAD) protein
~ ~ _ _ _
~~ ~ .'
~


NOV50b 220087646 1067 1_06_8 Human cadherin (CAD) protein
J ~


NOV50c 194877960 1069 1070 Human cadherin
(CAD) protein


NO_V_50d _ 1071u~~~ 1072 _
Y~~~ 192589297 x 'Human cadherin (CAD) protein
~ t


' CG58567-02 107_3_1074 Human cadherin (CAD) protein
N~OV50e _ _
~y


NOV50f CG58567-03 1075 ~ 1076 ~~Human cadherin (CAD) protein
~


34



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOVSOg CG58567 077 ,1 078 Human cadherin (CAD)
......_.. 04 1 _............_........._._............protein
............_.._.__,....... .......,....). .... ..,..._
..............._.,._..,.,.._.__._....._.....
.... .~,. ..,_, __.........._.....


NOVSOh CG58567-OS 079 ;1 080 Human..cadhenn (CAD)
a 1 _ _. _.. protein
___. _._ .. _ _......_._.. .. ._ _ _.... ...... ..
, _ _....,..__


NOVSOi 58567-06 081 1 082 rin (CAD) protein
1 Human cadhe
CG


NOVSla _ 083 ~ 084 _
~ CG59534-03~Y-1 Membrane glycoprotein
1 LIG-1 -


....___Homo Sapiens .__. __.
__ ._.... ......._......_......._.__......_........ .._. ....._......_
. _.. _.._..,..._.........._....._
_. .... .... ...........
. .


NOVSlb CG59534-Ol 085 1086 Membrane glycoprotein
~ 1 LIG-1 -


Homo Sapiens
_- ____ _._ -.. __ _ _ a ___
~


NOVSlc CG59534-02 1087 1088 Membrane glycoprotein
LIG-1 -


Homo_sapiens


NOVSld SNP133772551089 1090 Membrane glycoprotein
. LIG-1 -


_. _ __. . _ ~.._. _ Homo Sapiens _.


NOV52a CG59584-03 1091 1092 Ovostatin precursor
~


_._....... .... _.. ~Ovomacroglobulin)....'...callus
. ..._..._................__. gallus ....
_..
_ .
..


NOV52b CG59584-02 1093 1094 Ovostatinprecursor
~


_ ~ -~ _ (Ovomacroglobulin) -
~~ callus gallus


NOV52c 248210405 1095 1096 Ovostatin precursor
X ,


(Ovomacroglobulin) -
callus gallus


NOV52d 248210436 1097 1098 Ovostatin precursor
~ '


_ _ - ~.~ _ _ (Ovomacroglobulin) -_
~ ~ callus gallus
~


NOV52e 249357737 1099 1100 Ovostatin precursor
i ' (


Ovomacroglobulin).,..-.
~ Gallus,.gallus,...


NOV52f 248210430 1101 1102 Ovostatin precursor
x


_ ~ _-~ (Ovomacroglobulin) -
~ callus gallus


NOV52g 249357755 1103 1104 Ovostatin precursor


~ (Ovomacroglobulin) -
callus gallus


NOV52h CG59584-O1 1105 1106 Ovostatin precursor
~ ~


V ,~ __ ~. ~ ~ (Ovomacroglobulin),-
_ callus gallus
~


NOV53a CG56008-O1 1107 1108
' wJ


NOV54a CG59905-O1 1109 1110 CUB and sushi multiple
domains 1


_. ... .... .... ._.............. . protein.- .Iiomo. Sapiens..
..... .. ... .... ._ . ... .. .... .. ........_
. ....................._....... .. .. _..
... . . . .
..
....
..


NOV54b CG59905-03 1111 1112 CUB and sushi multiple
; domains 1


~ ~protein - Homo sap_iens
~


NOV54c 275631102 1113 1114 CUB and sushi multiple
domains 1


protein - Homo sapiens
Y


NOV54d CG59905-02 1115 1116 CUB and sushi multiple
; domains 1


_ a (protein - Hom_ o sapie_ns_
~


NOV54e ~SNP133825271117 1118 CUB and sushi multiple
= domains 1


_ _. _ _ ___... . . __. ..... Protein - Homo Sapiens.....
__.._. .. _ ._ . .._.. ...... ._ __.... ._.._ _ _
. _. ..._.._.. .._...... .._... . .._..___ .
......


NOV54f SNP133825201119 1120 CUB and sushi multiple
domains 1


_ _ _ _ ,__ protein - H_om__o sapie_ns
~ ~ __
~ .~


NOVSSa CG59932-O1 1121 1122_ _secreted protein sequ_e_nce
_ _ __ J ~


NOV56a CG92715-O1 1123 1124 secreted protein sequence
~ ~ ~ _


_ 1248576233 1125 1126 secreted protein sequence
NOV56b


NOV56c CG92715-02 1127 1128 secreted protein sequence





CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV56d CG92715-03 1129 ~ 1130 ~ 'secreted protein~sequence
_ .. i,~ .:.. .__ _ __.._.. _........._.. . . _... ~:.:._._~".
. ._ __.. _..... _._ . _._... ... ......
__. ,~ _


NOV57a CG92813-O1 1131 ~ 1132 Cadherin-related tumor suppressor
~


precursor (Fat protein) - Drosophila


melanogaster
_ ____._ ~ .._.~ .._ _. -._ __-


_.. ~ _.-_
NOV57b 260500971 1133 ~ 1134 ~Cadherin-related tumor suppressor


precursor (Fat protein) - Drosophila


_ melanogaster
~ ~ -y
~~


NOV57c 1135 31136
260500961 i Cadherin-related tumor suppressorw


precursor (Fat protein) - Drosophila


_ ~ melanogaste_r


NOV57d '258076331 1137 1138 iCadherin-related tumor suppressor


precursor (Fat protein) - Drosophila
~


_ ~ melanogaster
y


NOV57e '258076349 1139 1140 ~Cadherin-related tumor suppressor


precursor (Fat protein) - Drosophila


_ _ _ , melanogaster _ _
~ ~p
~ u~


NOV57f 1141 ' 1142
CG92813-02 Cadherin-related tumor suppressor


precursor (Fat protein) - Drosophila


_ ~ _ melanogaster
~ ~


NOV58a CG93387-O5 1143 1144 insulin-responsive sequence
DNA


_. .. . . .. _.. _ _. .. ~ ~bmding protein-1 -, Homo
.. .... . _ . __ .... sapiens .........
.
_.
.


NOV58b CG93387-Ol 1145 1146 insulin-responsive sequence
DNA


_ ._ _ ..____.____~ _ _ ~_,;Ibinding protein-1 -
H_omo sapiens _


NOVS8c CG93387-02 1147 1148 insulin-responsive sequence
DNA


binding protein-1 - Homo sapien_s
Y


NOV58d CG93387-03 1149 1150 ainsulin-responsive sequence
DNA


_ .. ~ ~ ~, binding protein-1 - Hom_o
_ Sapiens
Y


NOV58e CG93387-04 1151 1152 iinsulin-responsive sequence
' DNA


binding protein-1 - Homo Sapiens
_ _....~. _._.... _..........__.. ~. .. __..._....._ .~............
..._......... . ...._.. . ... ......................_..._....
. _ . _....


NOV58f SNP13382519 1153 1154 insulin-responsive sequence
DNA


_ binding protein-I -_ Homo Sapiens


NOV59a 'CG93871-O1 1155 1156 insulin-responsive sequence
DNA


binding protein-1 - Homo Sapiens


NOV59b ~ CG93871-OS 1157 1158 insulin-responsive sequence
DNA


_ ~ , _ binding protein-1 - Homo Sapiens
~


NOV59c 198488432 1159 1160
insulin-responsive sequence DNA


_ ... ....._.._................_..._'~'......__._~._..._.__ ._.__
....~~binding,protein-1.,..-.Homosapiens_
..._ __........
__._ .._..__.....__..


NOV59d ~ 198488424 1161 1162 insulin-responsive sequence
3 DNA


binding protein-1 - Homo Sapiens


NOV59e :198488428 1163 1164 insulin-responsive sequence
DNA


_ binding protein-1 - Homo Sapiens
. 1


NOV59f 1165 1166 insulin-responsive sequence
-198488440 DNA


binding protein-1 - H_omo Sapiens
w ~


NOV59g ~ CG93871-02 1167 1168 insulin-responsive sequence
' DNA


binding protein-1 - Homo Sapiens


36



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV59h CG93871-03 169 ; 1170 nsulin-responsive sequence
1 i DNA


_ binding protein-1 - Homo
sapiens


~NOV59i CG93871-04 1171 1172 insulin-responsive sequence
DNA


binding protein~_l -_Homo
sapie_ns


NOV60a CG94946-O1 1173 1174 AGRIN precursor - Homo
Sapiens


~ (Human), 2026 as


NOV60b 275631590 1175 1 I AGRIN precursor -Homo
~ 76. Sapiens
'


_ (Human), 2026 as
~~


NOV60c 275631564 1177 1178 AGRIN precursor - Homo
~ ' Sapiens


_..
v .._... ~Human), 2026 aa_ _ _
' . ........_....._._
l
~


omo sa dens
NOV60d ' 1179 1180 recursor -
CG94946-02 AGRIN p H p


_ (Human), 2026 aa_ _ _
__


NOV60e CG94946-03 1181 1182 AGRIN precursor - Homo
~ ~ sapiens


_ _ _ (Human), 2026 as _ ___
~


NOV60f CG94946-04 1183 1184 AGRIN precursor - Homo
~ ~ ~ ~ sapiens


_ ~ -I j (Human), 2026 as _


NOV60g CG94946-O5 1185 1186 AGKIN precursor - Homo
i ~ Sapiens


_ .... _ _..... ..._ ~H~an), 2026 as .. _ .
.~ _ _ ... .
~ ~


NOV60h CG94946-06 1187 1188 AGRIN precursor - Homo
Sapiens


_ _ __ __ _ _ _ (Human), 2026 as _ _ _
~ ~ ~ u


NOV60i CG94946-07 1189 1190 AGRIN precursor - Homo
~ f sapiens


~ (Hu_man), 2026 as


NOV6la - 1191 1192 colon cancer antigen protein
CG96384-O1 i
a


NOV6lb 277580745 1193 1194 colon cancer antigen protein


NOV6lc CG96384-02 1195 1196 colon cancer antigen protein


NOV_61 CG96384-03 1197 _ colon cancer antigen protein
d ! _: 1198
j


OV6la CG96384-04 1199 1200 colon cancer antigen prot_ein_
N ~


_ CG98011-O1 1201 1202 Neuronal transmembrane
NOV62a protein


_ S_litrk4 - Mus musculus
-~


NOV62b 192586956 1203 1204 Neuronal transmembrane
' protein


~ Slitrk4 - Mus musculus
~ ' ~


NOV62c 191999007 1205 1206 Neuronal transmembrane
protein


~ Slitrk4 - Mus musculus


_ _ _._._.__
NOV62d CG98011-02 1207 1208 Neuronal transmembrane
' protein


- Slitrk4 - Mus musculus
.


NOV62e CG98011-03 1209 1210 Neuronal transmembrane
protein


~~ ~Slitrk4 - Mus musculus


NOV62f _ 1211 1212 Neuronal transmembrane
CG98011-04 protein


~ Slitrk4 - Mus musculus


Table A indicates the homology of NOVX polypeptides to known protein families.
Thus, the nucleic acids and polypeptides, antibodies and related compounds
according to
the invention corresponding to a NOVX as identified in column 1 of Table A
will be useful
37



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in therapeutic and diagnostic applications implicated in, for example,
pathologies and
disorders associated with the known protein families identified in column 5 of
Table A.
Pathologies, diseases, disorders and condition and the like that are
associated with
NOVX sequences include, but are not limited to: e.g., cardiomyopathy,
atherosclerosis,
hypertension, congenital heart defects, aortic stenosis, atrial septal defect
(ASD), vascular
calcification, fibrosis, atrioventricular (A-V) canal defect, ductus
arteriosus, pulmonary
stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases,
tuberous
sclerosis, scleroderma, obesity, metabolic disturbances associated with
obesity,
transplantation, osteoarthritis, rheumatoid arthritis, osteochondrodysplasia,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer,
diabetes, metabolic
disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility,
glomerulonephritis, hemophilia, hypercoagulation, idiopathic thrombocytopenic
purpura,
immunodeficiencies, psoriasis, skin disorders, graft versus host disease,
AIDS, bronchial
asthma, lupus, Crohn's disease; inflammatory bowel disease, ulcerative
colitis, multiple
sclerosis, treatment of Albright Hereditary Ostoeodystrophy, infectious
disease, anorexia,
cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's
Disease,
Parkinson's Disorder, immune disorders, hematopoietic disorders, and the
various
dyslipidemias, schizophrenia, depression, asthma, emphysema, allergies, the
metabolic
syndrome X and wasting disorders associated with chronic diseases and various
cancers, as
well as conditions such as transplantation, neuroprotection, fertility, or
regeneration (irt
vitro and in vivo).
NOVX nucleic acids and their encoded polypeptides are useful in a variety of
applications and contexts. The various NOVX nucleic acids and polypeptides
according to
the invention are useful as novel members of the protein families according to
the presence
of domains and sequence relatedness to previously described proteins.
Additionally,
NOVX nucleic acids and polypeptides can also be used to identify proteins that
are
members of the family to which the NOVX polypeptides belong.
Consistent with other known members of the family of proteins, identified in
column 5 of Table A, the NOVX polypeptides of the present invention show
homology to,
and contain domains that are characteristic of, other members of such protein
families.
Details of the sequence relatedness and domain analysis for each NOVX are
presented in
Example A.
The NOVX nucleic acids and polypeptides can also be used to screen for
molecules,
which inhibit or enhance NOVX activity or function. Specifically, the nucleic
acids and
38



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polypeptides according to the invention may be used as targets for the
identification of
small molecules that modulate or inhibit diseases associated with the protein
families listed
in Table A.
The NOVX nucleic acids and polypeptides are also useful for detecting specific
cell
types. Details of the expression analysis for each NOVX are presented in
Example C.
Accordingly, the NOVX nucleic acids, polypeptides, antibodies and related
compounds
according to the invention will have diagnostic and therapeutic applications
in the detection
of a variety of diseases with differential expression in normal vs. diseased
tissues, e.g.
detection of a variety of cancers.
Additional utilities for NOVX nucleic acids and polypeptides according to the
invention are disclosed herein.
NOVX clones
NOVX nucleic acids and their encoded polypeptides are useful in a variety of
applications and contexts. The various NOVX nucleic acids and polypeptides
according to
the invention are useful as novel members of the protein families according to
the presence
of domains and sequence relatedness to previously described proteins.
Additionally,
NOVX nucleic acids and polypeptides can also be used to identify proteins that
are
members of the family to which the NOVX polypeptides belong.
The NOVX genes and their corresponding encoded proteins are useful for
preventing, treating or ameliorating medical conditions, e.g., by protein or
gene therapy.
Pathological conditions can be diagnosed by determining the amount of the new
protein in
a sample or by determining the presence of mutations in the new genes.
Specific uses are
described for each of the NOVX genes, based on the tissues in which they are
most highly
expressed. Uses include developing products for the diagnosis or treatment of
a variety of
diseases and disorders.
The NOVX nucleic acids and proteins of the invention are useful in potential
diagnostic and therapeutic applications and as a research tool. These include
serving as a
specific or selective nucleic acid or protein diagnostic and/or prognostic
marker, wherein
the presence or amount of the nucleic acid or the protein are to be assessed,
as well as
potential therapeutic applications such as the following: (i) a protein
therapeutic, (ii) a
small molecule drug target, (iii) an antibody target (therapeutic, diagnostic,
drug
targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy
(gene delivery/gene
39



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ablation), and (v) a composition promoting tissue regeneration in vitro and in
vivo (vi) a
biological defense weapon.
In one specific embodiment, the invention includes an isolated polypeptide
comprising an amino acid sequence selected from the group consisting of (a) a
mature
form of the amino acid sequence selected from the group consisting of SEQ m
NO: 2n,
wherein n is an integer between 1 and 606; (b) a variant of a mature form of
the amino acid
sequence selected from the group consisting of SEQ I~ NO: 2n, wherein n is an
integer
between 1 and 606, wherein any amino acid in the mature form is changed to a
different
amino acid, provided that no more than 15% of the amino acid residues in the
sequence of
the mature form are so changed; (c) an amino acid sequence selected from the
group
consisting of SEQ ll~ NO: 2n, wherein n is an integer between 1 and 606; (d) a
variant of
the amino acid sequence selected from the group consisting of SEQ ID N0:2n,
wherein n is
an integer between 1 and 606 wherein any amino acid specified in the chosen
sequence is
changed to a different amino acid, provided that no more than 15% of the amino
acid
residues in the sequence are so changed; and (e) a fragment of any of (a)
through (d).
In another specific embodiment, the invention includes an isolated nucleic
acid
molecule comprising a nucleic acid sequence encoding a polypeptide comprising
an amino
acid sequence selected from the group consisting of (a) a mature form of the
amino acid
sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 606; (b) a
variant of
a mature form of the amino acid sequence selected from the group consisting of
SEQ m
NO: 2n, wherein n is an integer between l and 606 wherein any amino acid in
the mature
form of the chosen sequence is changed to a different amino acid, provided
that no more
than 15% of the amino acid residues in the sequence of the mature form are so
changed; (c)
the amino acid sequence selected from the group consisting of SEQ m NO: 2n,
wherein n
is an integer between 1 and 606; (d) a variant of the amino acid sequence
selected from the
group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 606,
in which
any amino acid specified in the chosen sequence is changed to a different
amino acid,
provided that no more than 15% of the amino acid residues in the sequence are
so changed;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide
comprising the
amino acid sequence selected from the group consisting of SEQ m NO: 2n,
wherein n is an
integer between 1 and 606 or any variant of said polypeptide wherein any amino
acid of the
chosen sequence is changed to a different amino acid, provided that no more
than 10% of
the amino acid residues in the sequence are so changed; and (f) the complement
of any of
said nucleic acid molecules.



CA 02488547 2004-12-02
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In yet another specific embodiment, the invention includes an isolated nucleic
acid
molecule, wherein said nucleic acid molecule comprises a nucleotide sequence
selected
from the group consisting of (a) the nucleotide sequence selected from the
group
consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 606; (b)
a
nucleotide sequence wherein one or more nucleotides in the nucleotide sequence
selected
from the group consisting of SEQ m NO: 2n-l, wherein n is an integer between l
and 606
is changed from that selected from the group consisting of the chosen sequence
to a
different nucleotide provided that no more than 15% of the nucleotides are so
changed;
(c) a nucleic acid fragment of the sequence selected from the group consisting
of SEQ ID
NO: 2n-1, wherein n is an integer between 1 and 606; and (d) a nucleic acid
fragment
wherein one or more nucleotides in the nucleotide sequence selected from the
group
consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 606 is
changed
from that selected from the group consisting of the chosen sequence to a
different
nucleotide provided that no more than 15% of the nucleotides are so changed.
NOVX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that
encode
NOVX polypeptides or biologically active portions thereof. Also included in
the invention
are nucleic acid fragments sufficient for use as hybridization probes to
identify
NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR
primers for the amplification and/or mutation of NOVX nucleic acid molecules.
As used
herein, the term "nucleic acid molecule" is intended to include DNA molecules
(e.g.,
cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA
generated using nucleotide analogs, and derivatives, fragments and homologs
thereof. The
nucleic acid molecule may be single-stranded or double-stranded, but
preferably is
comprised double-stranded DNA.
A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a
"mature" form of a polypeptide or protein disclosed in the present invention
is the product
of a naturally occurring polypeptide or precursor form or proprotein. The
naturally
occurring polypeptide, precursor or proprotein includes, by way of nonlimiting
example,
the full-length gene product encoded by the corresponding gene. Alternatively,
it may be
defined as the polypeptide, precursor or proprotein encoded by an ORF
described herein.
The product "mature" form arises, by way of nonlimiting example, as a result
of one or
more naturally occurnng processing steps that may take place within the cell
(e.g., host
41



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cell) in which the gene product arises_ Examples of such processing steps
leading to a
"mature" form of a polypeptide or protein include the cleavage of the N-
terminal
methionine residue encoded by the initiation codon of an ORF, or the
proteolytic cleavage
of a signal peptide or leader sequence. Thus a mature form arising from a
precursor
S polypeptide or protein that has residues 1 to N, where residue 1 is the N-
terminal
methionine, would have residues 2 through N remaining after removal of the N-
terminal
methionine. Alternatively, a mature form arising from a precursor polypeptide
or protein
having residues 1 to N, in which an N-terminal signal sequence from residue 1
to residue M
is cleaved, would have the residues from residue M+1 to residue N remaining.
Further as
used herein, a "mature" form of a polypeptide or protein may arise from a step
of
post-translational modification other than a proteolytic cleavage event. Such
additional
processes include, by way of non-limiting example, glycosylation,
myristylation or
phosphorylation. In general, a mature polypeptide or protein may result from
the operation
of only one of these processes, or a combination of any of them.
The term "probe", as utilized herein, refers to nucleic acid sequences of
variable
length, preferably between at least about 10 nucleotides (nt), about 100 nt,
or as many as
approximately, e.g., 6,000 nt, depending upon the specific use. Probes are
used in the
detection of identical, similar, or complementary nucleic acid sequences.
Longer length
probes are generally obtained from a natural or recombinant source, are highly
specific, and
much slower to hybridize than shorter-length oligomer probes. Probes may be
single-
stranded or double-stranded and designed to have specificity in PCR, membrane-
based
hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as used herein, is a nucleic acid
that is
separated from other nucleic acid molecules which are present in the natural
source of the
nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences
which naturally
flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini 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 NOVX nucleic acid molecules can contain less
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 genomic DNA of the cell/tissue from which
the nucleic
acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an
"isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of other cellular
material, or
culture medium, or of chemical precursors or other chemicals.
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A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having
the
nucleotide sequence of SEQ >D NO:2n-1, wherein n is an integer between 1 and
606, or a
complement of this nucleotide sequence, can be isolated using standard
molecular biology
techniques and the sequence information provided herein. Using all or a
portion of the
S nucleic acid sequence of SEQ )D N0:2ra-1, wherein n is an integer between 1
and 606, as a
hybridization probe, NOVX molecules can be isolated using standard
hybridization and
cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR
CLONING: A
LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
NY, 199; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John
Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively, genomic DNA, as a template with appropriate oligonucleotide
primers
according to standard PCR amplification techniques. The nucleic acid so
amplified can be
cloned into an appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can
be
prepared by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues. A short oligonucleotide sequence may be based on, or designed from,
a genomic
or cDNA sequence and is used to amplify, confirm, or reveal the presence of an
identical,
similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides
comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in
length, preferably
about 15 nt to 30 nt in length. In one embodiment of the invention, an
oligonucleotide
comprising a nucleic acid molecule less than 100 nt in length would further
comprise at
least 6 contiguous nucleotides of SEQ ID N0:2n-1, wherein n is an integer
between l and
606, or a complement thereof. Oligonucleotides may be chemically synthesized
and may
also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a nucleic acid molecule that is a complement of the nucleotide
sequence shown
in SEQ >D N0:2fz-l, wherein n is an integer between 1 and 606, or a portion of
this
nucleotide sequence (e.g., a fragment that can be used as a probe or primer or
a fragment
encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid
molecule
that is complementary to the nucleotide sequence of SEQ ID N0:2n-1, wherein n
is an
integer between 1 and 606, is one that is sufficiently complementary to the
nucleotide
sequence of SEQ m N0:2n-l, wherein n is an integer between I and 606, that it
can
43



CA 02488547 2004-12-02
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hydrogen bond with few or no mismatches to the nucleotide sequence shown in
SEQ m
N0:2n-l, wherein n is an integer between l and 606, thereby forming a stable
duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base pairing between nucleotides units of a nucleic acid molecule, and the
term "binding"
means the physical or chemical interaction between two polypeptides or
compounds or
associated polypeptides or compounds or combinations thereof. Binding includes
ionic,
non-ionic, van der Waals, hydrophobic interactions, and the like. A physical
interaction
can be either direct or indirect. Indirect interactions may be through or due
to the effects of
another polypeptide or compound. Direct binding refers to interactions that do
not take
place through, or due to, the effect of another polypeptide or compound, but
instead are
without other substantial chemical intermediates.
A "fragment" provided herein is defined as a sequence of at least 6
(contiguous)
nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to
allow for specific
hybridization in the case of nucleic acids or for specific recognition of an
epitope in the
case of amino acids, and is at most some portion less than a full length
sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or
amino acid
sequence of choice.
A full-length NOVX clone is identified as containing an ATG translation start
codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence
lacking an
ATG start codon therefore encodes a truncated C-terminal fragment of the
respective
NOVX polypeptide, and requires that the corresponding full-length cDNA extend
in the 5'
direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence
lacking an
in-frame stop codon similarly encodes a truncated N-terminal fragment of the
respective
NOVX polypeptide, and requires that the corresponding full-length cDNA extend
in the 3'
direction of the disclosed sequence.
A "derivative" is a nucleic acid sequence or amino acid sequence formed from
the
native compounds either directly, by modification or partial substitution. An
"analog" is a
nucleic acid sequence or amino acid sequence that has a structure similar to,
but not
identical to, the native compound, e.g. they differs from it in respect to
certain components
or side chains. Analogs may be synthetic or derived from a different
evolutionary origin
and may have a similar or opposite metabolic activity compared to wild type. A
"homolog" is a nucleic acid sequence or amino acid sequence of a particular
gene that is
derived from different species.
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Derivatives and analogs may be full length or other than full length.
Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not
limited to,
molecules comprising regions that are substantially homologous to the nucleic
acids or
proteins of the invention, in various embodiments, by at least about 70%, 80%,
or 95%
S identity (with a preferred identity of 80-95%) over a nucleic acid or amino
acid sequence of
identical size or when compared to an aligned sequence in which the alignment
is done by a
computer homology program known in the art, or whose encoding nucleic acid is
capable
of hybridizing to the complement of a sequence encoding the proteins under
stringent,
moderately stringent, or low stringent conditions. See e.g. Ausubel, et al.,
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and
below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences include
those
sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed
in
different tissues of the same organism as a result of, for example,
alternative splicing of
RNA. Alternatively, isoforms can be encoded by different genes. In the
invention,
homologous nucleotide sequences include nucleotide sequences encoding for a
NOVX
polypeptide of species other than humans, including, but not limited to:
vertebrates, and
thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and
other organisms.
Homologous nucleotide sequences also include, but are not limited to,
naturally occurring
allelic variations and mutations of the nucleotide sequences set forth herein.
A homologous
nucleotide sequence does not, however, include the exact nucleotide sequence
encoding
human NOVX protein. Homologous nucleic acid sequences include those nucleic
acid
sequences that encode conservative amino acid substitutions (see below) in SEQ
ID
N0:2n-1, wherein n is an integer between l and 606, as well as a polypeptide
possessing
NOVX biological activity. Various biological activities of the NOVX proteins
are
described below.
A NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could
potentially be
translated into a polypeptide. A stretch of nucleic acids comprising an ORF is
uninterrupted by a stop codon. An ORF that represents the coding sequence for
a full
protein begins with an ATG "start" codon and terminates with one of the three
"stop"
codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF
may be



CA 02488547 2004-12-02
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any part of a coding sequence, with or without a start codon, a stop codon, or
both. For an
ORF to be considered as a good candidate for coding for a bona fide cellular
protein, a
minimum size requirement is often set, e.g., a stretch of DNA that would
encode a protein
of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes
allows for the generation of probes and primers designed for use in
identifying and/or
cloning NOVX homologues in other cell types, e.g. from other tissues, as well
as NOVX
homologues from other vertebrates. The probelprimer typically comprises
substantially
purified oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide
sequence that hybridizes under stringent conditions to at least about 12, 25,
50,100, 150,
200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ
)D
N0:2n-1, wherein n is an integer between 1 and 606; or an anti-sense strand
nucleotide
sequence of SEQ )D N0:2n-l, wherein n is an integer between 1 and 606; or of a
naturally
occurnng mutant of SEQ m N0:2n-1, wherein n is an integer between 1 and 606.
Probes based on the human NOVX nucleotide sequences can be used to detect
transcripts or genomic sequences encoding the same or homologous proteins. In
various
embodiments, the probe has a detectable label attached, e.g. the label can be
a radioisotope,
a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be
used as a
part of a diagnostic test kit for identifying cells or tissues which rnis-
express a NOVX
protein, such as by measuring a level of a NOVX-encoding nucleic acid in a
sample of cells
from a subject e.g., detecting NOVX mRNA levels or determining whether a
genomic
NOVX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of a NOVX polypeptide"
refers
to polypeptides exhibiting activity similar, but not necessarily identical to,
an activity of a
polypeptide of the invention, including mature forms, as measured in a
particular biological
assay, with or without dose dependency. A nucleic acid fragment encoding a
"biologically-active portion of NOVX" can be prepared by isolating a portion
of SEQ ID
N0:2n-l, wherein n is an integer between 1 and 606, that encodes a polypeptide
having a
NOVX biological activity (the biological activities of the NOVX proteins are
described
below), expressing the encoded portion of NOVX protein (e.g., by recombinant
expression
in vitro) and assessing the activity of the encoded portion of NOVX.
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NOVX Single Nucleotide Polymorphisms
Variant sequences are also included in this application. A variant sequence
can
include a single nucleotide polymorphism (SNP). A SNP can, in some instances,
be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP
originates as a cDNA. A SNP can arise in several ways. For example, a SNP may
be due to
a substitution of one nucleotide for another at the polymorphic site. Such a
substitution can
be either a transition or a transversion. A SNP can also arise from a deletion
of a
nucleotide or an insertion of a nucleotide, relative to a reference allele. In
this case, the
polymorphic site is a site at which one allele bears a gap with respect to a
particular
nucleotide in another allele. SNPs occurring within genes may result in an
alteration of the
amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may
also be
silent, when a codon including a SNP encodes the same amino acid as a result
of the
redundancy of the genetic code. SNPs occurnng outside the region of a gene, or
in an
intron within a gene, do not result in changes in any amino acid sequence of a
protein but
may result in altered regulation of the expression pattern. Examples include
alteration in
temporal expression, physiological response regulation, cell type expression
regulation,
intensity of expression, and stability of transcribed message.
SeqCalling assemblies produced by the exon linking process were selected and
extended using the following criteria. Genomic clones having regions with 98%
identity to
all or part of the initial or extended sequence were identified by BLASTN
searches using
the relevant sequence to query human genomic databases. The genomic clones
that
resulted were selected for further analysis because this identity indicates
that these clones
contain the genomic locus for these SeqCalling assemblies. These sequences
were
analyzed for putative coding regions as well as for similarity to the known
DNA and
protein sequences. Programs used for these analyses include Grail, Genscan,
BLAST,
HMMER, FASTA, Hybrid and other relevant programs.
Some additional genomic regions may have also been identified because selected
SeqCalling assemblies map to those regions. Such SeqCalling sequences may have
overlapped with regions defined by homology or exon prediction. They may also
be
included because the location of the fragment was in the vicinity of genomic
regions
identified by similarity or exon prediction that had been included in the
original predicted
sequence. The sequence so identified was manually assembled and then may have
been
extended using one or more additional sequences taken from CuraGen
Corporation's human
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SeqCalling database. SeqCalling fragments suitable for inclusion were
identified by the
CuraTools~ program SeqExtend or by identifying SeqCalling fragments mapping to
the
appropriate regions of the genomic clones analyzed.
The regions defined by the procedures described above were then manually
integrated and corrected for apparent inconsistencies that may have arisen,
for example,
from miscalled bases in the original fragments or from discrepancies between
predicted
exon junctions, EST locations and regions of sequence similarity, to derive
the final
sequence disclosed herein. When necessary, the process to identify and analyze
SeqCalling
assemblies and genomic clones was reiterated to derive the full length
sequence (Alderborn
et al., Determination of Single Nucleotide Polymorphisms by Real-time
Pyrophosphate
DNA Sequencing. Genome Research. 10 (8) 1249-1265, 2000).
Variants are reported individually but any combination of all or a select
subset of
variants are also included as contemplated NOVX embodiments of the invention.
NOVX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequences of SEQ m N0:2n-1, wherein n is an integer between 1 and
606, due
to degeneracy of the genetic code and thus encode the same NOVX proteins as
that
encoded by the nucleotide sequences of SEQ m N0:2n-l, wherein n is an integer
between
l and 606. In another embodiment, an isolated nucleic acid molecule of the
invention has a
nucleotide sequence encoding a protein having an amino acid sequence of SEQ >D
N0:2n,
wherein n is an integer between 1 and 606.
In addition to the human NOVX nucleotide sequences of SEQ 1D N0:2n-l, wherein
n is an integer between 1 and 606, it will be appreciated by those skilled in
the art that
DNA sequence polymorphisms that lead to changes in the amino acid sequences of
the
NOVX polypeptides may exist within a population (e.g., the human population).
Such
genetic polymorphism in the NOVX genes may exist among individuals within a
population due to natural allelic variation. As used herein, the terms "gene"
and
"recombinant gene" refer to nucleic acid molecules comprising an open reading
frame
(ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such
natural
allelic variations can typically result in 1-5% variance in the nucleotide
sequence of the
NOVX genes. Any and all such nucleotide variations and resulting amino acid
polymorphisms in the NOVX polypeptides, which are the result of natural
allelic variation
48



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
and that do not alter the functional activity of the NOVX polypeptides, are
intended to be
within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species,
and
thus that have a nucleotide sequence that differs from a human SEQ ID N0:2n-l,
wherein n
is an integer between 1 and 606, are intended to be within the scope of the
invention.
Nucleic acid molecules corresponding to natural allelic variants and
homologues of the
NOVX cDNAs of the invention can be isolated based on their homology to the
human
NOVX nucleic acids disclosed herein using the human cDNAs, or a portion
thereof, as a
hybridization probe according to standard hybridization techniques under
stringent
hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 6 nucleotides in length and hybridizes under stringent
conditions to the
nucleic acid molecule comprising the nucleotide sequence of SEQ )D NO:2n-1,
wherein n
is an integer between 1 and 606. In another embodiment, the nucleic acid is at
least 10, 25,
50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In
yet another
embodiment, an isolated nucleic acid molecule of the invention hybridizes to
the coding
region. As used herein, the term "hybridizes under stringent conditions" is
intended to
describe conditions for hybridization and washing under which nucleotide
sequences at
least about 65% homologous to each other typically remain hybridized to each
other.
Homologs (i.e., nucleic acids encoding NOVX proteins derived from species
other
than human) or other related sequences (e.g., paralogs) can be obtained by
low, moderate or
high stringency hybridization with all or a portion of the particular human
sequence as a
probe using methods well known in the art for nucleic acid hybridization and
cloning.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to
no other sequences. Stringent conditions are sequence-dependent and will be
different in
different circumstances. Longer sequences hybridize specifically at higher
temperatures
than shorter sequences. Generally, stringent conditions are selected to be
about 5 °C lower
than the thermal melting point (Tm) for the specific sequence at a defined
ionic strength
and pH. The Tm is the temperature (under defined ionic strength, pH and
nucleic acid
concentration) at which 50% of the probes complementary to the target sequence
hybridize
to the target sequence at equilibrium. Since the target sequences are
generally present at
excess, at Tm, 50% of the probes are occupied at equilibrium. Typically,
stringent
conditions will be those in which the salt concentration is Less than about
1.0 M sodium ion,
49



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WO 03/102155 PCT/US03/17430
typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to ~.3 and
the
temperature is at least about 30 °C for short probes, primers or
oligonucleotides (e.g., 10 nt .
to 50 nt) and at least about 60 °C for longer probes, primers and
oligonucleotides.
Stringent conditions may also be achieved with the addition of destabilizing
agents, such as
formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons,
N.Y. (199), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at
least about
65%, 70%, 75%, ~5%, 90%, 95%, 9~%, or 99% homologous to each other typically
remain
hybridized to each other. A non-limiting example of stringent hybridization
conditions are
hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm
DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at
50°C. An
isolated nucleic acid molecule of the invention that hybridizes under
stringent conditions to
a sequence of SEQ m N0:2~a-1, wherein n is an integer between 1 and 606,
corresponds to
a naturally-occurring nucleic acid molecule. As used herein, a "naturally-
occurnng"
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 a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of SEQ m NO:2n-1, wherein n
is an
integer between 1 and 606, or fragments, analogs or derivatives thereof, under
conditions of
moderate stringency is provided. A non-limiting example of moderate stringency
hybridization conditions are hybridization in 6X SSC, SX Reinhardt's solution,
0.5% SDS
and 100 mg/ml denatured salmon sperm DNA at 55 °C, followed by one or
more washes in
1X SSC, 0.1 % SDS at 37 °C. Other conditions of moderate stringency
that may be used
are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993,
CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER
AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule comprising the nucleotide sequences of SEQ m N0:2n-l, wherein n is an
integer
between l and 606, or fragments, analogs or derivatives thereof, under
conditions of low
stringency, is provided. A non-limiting example of low stringency
hybridization conditions
are hybridization in 35% formamide, SX SSC, SO mM Tris-HCl (pH 7.5), 5 mM
EDTA,
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%



CA 02488547 2004-12-02
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(wtlvol) dextran sulfate at 40°C, followed by one or more washes in 2X
SSC, 25 mM
Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions
of low
stringency that may be used are well known in the art (e.g., as employed for
cross-species
hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, NY, and I~riegler, 1990, GENE TRANSFER
AND
EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981.
Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurnng allelic variants of NOVX sequences that may
exist in the population, the skilled artisan will further appreciate that
changes can be
introduced by mutation into the nucleotide sequences of SEQ m N0:2n-l, wherein
n is an
integer between 1 and 606, thereby leading to changes in the amino acid
sequences of the
encoded NOVX protein, without altering the functional ability of that NOVX
protein. For
example, nucleotide substitutions leading to amino acid substitutions at "non-
essential"
amino acid residues can be made in the sequence of SEQ )D N0:2rr, wherein n is
an integer
between 1 and 606. A "non-essential" amino acid residue is a residue that can
be altered
from the wild-type sequences of the NOVX proteins without altering their
biological
activity, whereas an "essential" amino acid residue is required for such
biological activity.
For example, amino acid residues that are conserved among the NOVX proteins of
the
invention are predicted to be particularly non-amenable to alteration. Amino
acids for
which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding
NOVX
proteins that contain changes in amino acid residues that are not essential
for activity. Such
NOVX proteins differ in amino acid sequence from SEQ ID N0:2n-1, wherein n is
an
integer between l and 606, yet retain biological activity. In one embodiment,
the isolated
nucleic acid molecule comprises a nucleotide sequence encoding a protein,
wherein the
protein comprises an amino acid sequence at least about 40% homologous to the
amino
acid sequences of SEQ ID N0:2n, wherein n is an integer between 1 and 606.
Preferably,
the protein encoded by the nucleic acid molecule is at least about 60%
homologous to SEQ
1D N0:2ra, wherein n is an integer between 1 and 606; more preferably at least
about 70%
homologous to SEQ m N0:2rr, wherein n is an integer between 1 and 606; still
more
preferably at least about 80% homologous to SEQ m NO:2n, wherein n is an
integer
between 1 and 606; even more preferably at least about 90% homologous to SEQ m
S1



CA 02488547 2004-12-02
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N0:2n, wherein n is an integer between l and 606; and most preferably at least
about 95%
homologous to SEQ ID N0:2n, wherein n is an integer between l and 606.
An isolated nucleic acid molecule encoding a NOVX protein homologous to the
protein of SEQ ID N0:2n, wherein n is an integer between 1 and 606, can be
created by
introducing one or more nucleotide substitutions, additions or deletions into
the nucleotide
sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 606, such
that one or
more amino acid substitutions, additions or deletions are introduced into the
encoded
protein.
Mutations can be introduced any one of SEQ ID N0:2n-1, wherein n is an integer
between l and 606, by standard techniques, such as site-directed mutagenesis
and
PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions
are made at
one or more predicted, non-essential amino acid residues. A "conservative
amino acid
substitution" is one in which the amino acid residue is replaced with an amino
acid residue
having a similar side chain. Families of amino acid residues having similar
side chains
have been defined within the art. These families include amino acids with
basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid),
uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine).
Thus, a predicted non-essential amino acid residue in the NOVX protein is
replaced with
another amino acid residue from the same side chain family. Alternatively, in
another
embodiment, mutations can be introduced randomly along all or part of a NOVX
coding
sequence, such as by saturation mutagenesis, and the resultant mutants can be
screened for
NOVX biological activity to identify mutants that retain activity. Following
mutagenesis
of a nucleic acid of SEQ ID N0:2zz-1, wherein n is an integer between 1 and
606, the
encoded protein can be expressed by any recombinant technology known in the
art and the
activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side
chain
interactions. Substituted amino acids may be fully conserved "strong" residues
or fully
conserved "weak" residues. The "strong" group of conserved amino acid residues
may be
any one of the following groups: STA, NEQI~, NHQK, NDEQ, QHRK, MILV, MILF, HY,
FYW, wherein the single letter amino acid codes are grouped by those amino
acids that
may be substituted for each other. Likewise, the "weak" group of conserved
residues may
52



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQI~,
N~EQHK, NEQHRK, HFY, wherein the letters within each group represent the
single
letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to
form protein:protein interactions with other NOVX proteins, other cell-surface
proteins, or
biologically-active portions thereof, (ii) complex formation between a mutant
NOVX
protein and a NOVX ligand; or (iii) the ability of a mutant NOVX protein to
bind to an
intracellular target protein or biologically-active portion thereof; (e.g.
avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the
ability
to regulate a specific biological function (e.g., regulation of insulin
release).
Interfering RNA
In one aspect of the invention, NOVX gene expression can be attenuated by RNA
interference. One approach well-known in the art is short interfering RNA
(siRNA)
mediated gene silencing where expression products of a NOVX gene are targeted
by
specific double stranded NOVX derived siRNA nucleotide sequences that are
complementary to at least a 19-25 nt long segment of the NOVX gene transcript,
including
the 5' untranslated (IJT) region, the ORF, or the 3' UT region. See, e.g., PCT
applications
WO00/44895, W099/32619, WO01/75164, WO01/92513, WO 01/29058, WO01/89304,
W002/16620, and W002/29858, each incorporated by reference herein in their
entirety.
Targeted genes can be a NOVX gene, or an upstream or downstream modulator of
the
NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX
gene include, e.g., a transcription factor that binds the NOVX gene promoter,
a kinase or
phosphatase that interacts with a NOVX polypeptide, and polypeptides involved
in a
NOVX regulatory pathway.
According to the methods of the present invention, NOVX gene expression is
silenced using short interfering RNA. A NOVX polynucleotide according to the
invention
includes a siRNA polynucleotide. Such a NOVX siRNA can be obtained using a
NOVX
polynucleotide sequence, for example, by processing the NOVX
ribopolynucleotide
sequence in a cell-free system, such as but not limited to a Drosophila
extract, or by
transcription of recombinant double stranded NOVX RNA or by chemical synthesis
of
nucleotide sequences homologous to a NOVX sequence. See, e.g., Tuschl, Zamore,
Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated
herein by
reference in its entirety. When synthesized, a typical 0.2 micromolar-scale
RNA synthesis
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CA 02488547 2004-12-02
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provides about 1 milligram of siRNA, which is sufficient for 1000 transfection
experiments
using a 24-well tissue culture plate format.
The most efficient silencing is generally observed with siRNA duplexes
composed
of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to
have a 2-nt
S 3' overhang. The sequence of the 2-nt 3' overhang makes an additional small
contribution
to the specificity of siRNA target recognition. The contribution to
specificity is localized to
the unpaired nucleotide adjacent to the first paired bases. In one embodiment,
the
nucleotides in the 3' overhang are ribonucleotides. In an alternative
embodiment, the
nucleotides in the 3' overhang are deoxyribonucleotides. Using 2'-
deoxyribonucleotides in
the 3' overhangs is as efficient as using ribonucleotides, but
deoxyribonucleotides are often
cheaper to synthesize and are most likely more nuclease resistant.
A contemplated recombinant expression vector of the invention comprises a NOVX
DNA molecule cloned into an expression vector comprising operatively-linked
regulatory
sequences flanking the NOVX sequence in a manner that allows for expression
(by
transcription of the DNA molecule) of both strands. An RNA molecule that is
antisense to
NOVX mRNA is transcribed by a first promoter (e.g., a promoter sequence 3' of
the cloned
DNA) and an RNA molecule that is the sense strand for the NOVX mRNA is
transcribed
by a second promoter (e.g., a promoter sequence 5' of the cloned DNA). The
sense and
antisense strands may hybridize in vivo to generate siRNA constructs for
silencing of the
NOVX gene. Alternatively, two constructs can be utilized to create the sense
and
anti-sense strands of a siRNA construct. Finally, cloned DNA can encode a
construct
having secondary structure, wherein a single transcript has both the sense and
complementary antisense sequences from the target gene or genes. In an example
of this
embodiment, a hairpin RNAi product is homologous to all or a portion of the
target gene.
In another example, a hairpin RNAi product is a siRNA. The regulatory
sequences
flanking the NOVX sequence may be identical or may be different, such that
their
expression may be modulated independently, or in a temporal or spatial manner.
In a specific embodiment, siRNAs are transcribed intracellularly by cloning
the
NOVX gene templates into a vector containing, e.g., a RNA pol III
transcription unit from
the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA Hl. One example of
a
vector system is the GeneSuppressor~ RNA Interference kit (commercially
available from
Imgenex). The U6 and H1 promoters are members of the type III class of Pol III
promoters.
The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1
for Hl
promoters is adenosine. The termination signal for these promoters is defined
by five
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CA 02488547 2004-12-02
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consecutive thymidines. The transcript is typically cleaved after the second
uridine.
Cleavage at this position generates a 3' UU overhang in the expressed siRNA,
which is
similar to the 3' overhangs of synthetic siRNAs. Any sequence less than 400
nucleotides in
length can be transcribed by these promoter, therefore they are ideally suited
for the
expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-
nucleotide RNA
stem-loop transcript.
A siRNA vector appears to have an advantage over synthetic siRNAs where long
term knock-down of expression is desired. Cells transfected with a siRNA
expression
vector would experience steady, long-term mRNA inhibition. W contrast, cells
transfected
with exogenous synthetic siRNAs typically recover from mRNA suppression within
seven
days or ten rounds of cell division. The long-term gene silencing ability of
siRNA
expression vectors may provide for applications in gene therapy.
In general, siRNAs are chopped from longer dsRNA by an ATP-dependent
ribonuclease called DICER. DICER is a member of the RNase III family of
double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular
proteins
into an endonuclease complex. In vitro studies in Drosophila suggest that the
siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex,
called
an RNA-induced silencing complex (RISC), which contains an endoribonuclease
that is
distinct from DICER. RISC uses the sequence encoded by the antisense siRNA
strand to
fnd and destroy mRNAs of complementary sequence. The siRNA thus acts as a
guide,
restricting the ribonuclease to cleave only mRNAs complementary to one of the
two siRNA
strands.
A NOVX mRNA region to be targeted by siRNA is generally selected from a
desired NOVX sequence beginning 50 to 100 nt downstream of the start codon.
Alternatively, 5' or 3' UTRs and regions nearby the start codon can be used
but are
generally avoided, as these may be richer in regulatory protein binding sites.
UTR-binding
proteins and/or translation initiation complexes may interfere with binding of
the siRNP or
RISC endonuclease complex. An initial BLAST homology search for the selected
siRNA
sequence is done against an available nucleotide sequence library to ensure
that only one
gene is targeted. Specificity of target recognition by siRNA duplexes indicate
that a single
point mutation located in the paired region of an siRNA duplex is sufficient
to abolish
target mRNA degradation. See, Elbashir et al. 2001 EMBO J. 20(23):6877-88.
Hence,
consideration should be taken to accommodate SNPs, polymorphisms, allelic
variants or
species-specific variations when targeting a desired gene.



CA 02488547 2004-12-02
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In one embodiment, a complete NOVX siRNA experiment includes the proper
negative control. A negative control siRNA generally has the same nucleotide
composition
as the NOVX siRNA but lack significant sequence homology to the genome.
Typically,
one would scramble the nucleotide sequence of the NOVX siRNA and do a homology
search to make sure it lacks homology to any other gene.
Two independent NOVX siRNA duplexes can be used to knock-down a target
NOVX gene. This helps to control for specificity of the silencing effect. In
addition,
expression of two independent genes can be simultaneously knocked down by
using equal
concentrations of different NOVX siRNA duplexes, e.g., a NOVX siRNA and an
siRNA
for a regulator of a NOVX gene or polypeptide. Availability of siRNA-
associating proteins
is believed to be more limiting than target mRNA accessibility.
A targeted NOVX region is typically a sequence of two adenines (AA) and two
thymidines (TT) divided by a spacer region of nineteen (N19) residues (e.g.,
AA(N19)TT).
A desirable spacer region has a GlC-content of approximately 30% to 70%, and
more
preferably of about 50%. If the sequence AA(N19)TT is not present in the
target sequence,
an alternative target region would be AA(N21). The sequence of the NOVX sense
siRNA
corresponds to (N19)TT or N21, respectively. In the latter case, conversion of
the 3' end of
the sense siRNA to TT can be performed if such a sequence does not naturally
occur in the
NOVX polynucleotide. The rationale for this sequence conversion is to generate
a
symmetric duplex with respect to the sequence composition of the sense and
antisense 3'
overhangs. Symmetric 3' overhangs may help to ensure that the siRNPs are
formed with
approximately equal ratios of sense and antisense target RNA-cleaving siRNPs.
See, e.g.,
Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200, incorporated
by
reference herein in its entirely. The modification of the overhang of the
sense sequence of
the siRNA duplex is not expected to affect targeted mRNA recognition, as the
antisense
siRNA strand guides target recognition.
Alternatively, if the NOVX target mRNA does not contain a suitable AA(N21)
sequence, one may search for the sequence NA(N21). Further, the sequence of
the sense
strand and antisense strand may still be synthesized as 5' (N19)TT, as it is
believed that the
sequence of the 3'-most nucleotide of the antisense siRNA does not contribute
to
specificity. Unlike antisense or ribozyme technology, the secondary structure
of the target
mRNA does not appear to have a strong effect on silencing. See, Harborth, et
al. (2001) J.
Cell Science 114: 4557-4565, incorporated by reference in its entirety.
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CA 02488547 2004-12-02
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Transfection of NOVX siRNA duplexes can be achieved using standard nucleic
acid transfection methods, for example, OLIGOFECTAMINE Reagent (commercially
available from Invitrogen). An assay for NOVX gene silencing is generally
performed
approximately 2 days after transfection. No NOVX gene silencing has been
observed in
the absence of transfection reagent, allowing for a comparative analysis of
the wild-type
and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-
well plate,
approximately 0.84 ~,g of the siRNA duplex is generally sufficient. Cells are
typically
seeded the previous day, and are transfected at about 50% confluence. The
choice of cell
culture media and conditions are routine to those of skill in the art, and
will vary with the
choice of cell type. The efficiency of transfection may depend on the cell
type, but also on
the passage number and the confluency of the cells. The time and the manner of
formation
of siRNA-liposome complexes (e.g. inversion versus vortexing) are also
critical. Low
transfection efficiencies are the most frequent cause of unsuccessful NOVX
silencing. The
efficiency of transfection needs to be carefully examined for each new cell
line to be used.
Preferred cell are derived from a mammal, more preferably from a rodent such
as a rat or
mouse, and most preferably from a human. Where used for therapeutic treatment,
the cells
are preferentially autologous, although non-autologous cell sources are also
contemplated
as within the scope of the present invention.
For a control experiment, transfection of 0.84 ~g single-stranded sense NOVX
siRNA will have no effect on NOVX silencing, and 0.84 ~g antisense siRNA has a
weak
silencing effect when compared to 0.84 ~.g of duplex siRNAs. Control
experiments again
allow for a comparative analysis of the wild-type and silenced NOVX
phenotypes. To
control for transfection efficiency, targeting of common proteins is typically
performed, for
example targeting of lamin A/C or transfection of a CMV-driven EGFP-expression
plasmid
(e.g. commercially available from Clontech). In the above example, a
determination of the
fraction of lamin AlC knockdown in cells is determined the next day by such
techniques as
immunofluorescence, Western blot, Northern blot or other similar assays for
protein
expression or gene expression. Lamin A/C monoclonal antibodies may be obtained
from
Santa Cruz Biotechnology.
Depending on the abundance and the half life (or turnover) of the targeted
NOVX
polynucleotide in a cell, a knock-down phenotype may become apparent after 1
to 3 days,
or even later. In cases where no NOVX knock-down phenotype is observed,
depletion of
the NOVX polynucleotide may be observed by immunofluorescence or Western
blotting.
If the NOVX polynucleotide is still abundant after 3 days, cells need to be
split and
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CA 02488547 2004-12-02
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transferred to a fresh 24-well plate for re-transfection. If no knock-down of
the targeted
protein is observed, it may be desirable to analyze whether the target mRNA
(NOVX or a
NOVX upstream or downstream gene) was effectively destroyed by the transfected
siRNA
duplex. Two days after transfection, total RNA is prepared, reverse
transcribed using a
target-specific primer, and PCR-amplified with a primer pair covering at least
one
exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR
of a
non-targeted mRNA is also needed as control. Effective depletion of the mRNA
yet
undetectable reduction of target protein may indicate that a large reservoir
of stable NOVX
protein may exist in the cell. Multiple transfection in sufficiently long
intervals may be
necessary until the target protein is finally depleted to a point where a
phenotype may
become apparent. If multiple transfection steps are required, cells are split
2 to 3 days after
transfection. The cells may be transfected immediately after splitting.
An inventive therapeutic method of the invention contemplates administering a
NOVX siRNA construct as therapy to compensate for increased or aberrant NOVX
expression or activity. The NOVX ribopolynucleotide is obtained and processed
into
siRNA fragments, or a NOVX siRNA is synthesized, as described above. The NOVX
siRNA is administered to cells or tissues using known nucleic acid
transfection techniques,
as described above. A NOVX siRNA specific for a NOVX gene will decrease or
knockdown NOVX transcription products, which will lead to reduced NOVX
polypeptide
production, resulting in reduced NOVX polypeptide activity in the cells or
tissues.
The present invention also encompasses a method of treating a disease or
condition
associated with the presence of a NOVX protein in an individual comprising
administering
to the individual an RNAi construct that targets the mRNA of the protein (the
mRNA that
encodes the protein) for degradation. A specific RNAi construct includes a
siRNA or a
double stranded gene transcript that is processed into siRNAs. Upon treatment,
the target
protein is not produced or is not produced to the extent it would be in the
absence of the
treatment.
Where the NOVX gene function is not correlated with a known phenotype, a
control sample of cells or tissues from healthy individuals provides a
reference standard for
determining NOVX expression levels. Expression levels are detected using the
assays
described, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the
like. A
subject sample of cells or tissues is taken from a mammal, preferably a human
subject,
suffering from a disease state. The NOVX ribopolynucleotide is used to produce
siRNA
constructs, that are specific for the NOVX gene product. These cells or
tissues are treated
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by administering NOVX siRNA's to. the cells or tissues by methods described
for the
transfection of nucleic acids into a cell or tissue, and a change in NOVX
polypeptide or
polynucleotide expression is observed in the subject sample relative to the
control sample,
using the assays described. This NOVX gene knockdown approach provides a rapid
method for determination of a NOVX minus (NOVX~ phenotype in the treated
subject
sample. The NOVX- phenotype observed in the treated subj ect sample thus
serves as a
marker for monitoring the course of a disease state during treatment.
In specific embodiments, a NOVX siRNA is used in therapy. Methods for the
generation and use of a NOVX siRNA are known to those skilled in the art.
Example
techniques are provided below.
Production of RNAs
Sense RNA (ssRNA) and antisense RNA (asRNA) of NOVX are produced using
known methods such as transcription in RNA expression vectors. In the initial
experiments, the sense and antisense RNA are about 500 bases in length each.
The
produced ssRNA and asRNA (0.5 ~,M) in 10 mM Tris-HCI (pH 7.5) with 20 mM NaCI
were heated to 95° C for 1 min then cooled and annealed at room
temperature for 12 to 16
h. The RNAs are precipitated and resuspended in lysis buffer (below). To
monitor
annealing, RNAs are electrophoresed in a 2% agarose gel in TBE buffer and
stained with
ethidium bromide. See, e.g., Sambrook et al., Molecular Cloning. Cold Spring
Harbor
Laboratory Press, Plainview, N.Y. (1989).
Lysate Preparation
Untreated rabbit reticulocyte lysate (Ambion) are assembled according to the
manufacturer's directions. dsRNA is~incubated in the lysate at 30° C
for 10 min prior to the
addition of mRNAs. Then NOVX mRNAs are added and the incubation continued for
an
additional 60 min. The molar ratio of double stranded RNA and mRNA is about
200:1.
The NOVX mRNA is radiolabeled (using known techniques} and its stability is
monitored
by gel electrophoresis.
In a parallel experiment made with the same conditions, the double stranded
RNA is
internally radiolabeled with a 32P-ATP. Reactions are stopped by the addition
of 2 X
proteinase I~ buffer and deproteinized as described previously (Tuschl et al.,
Genes Dev.,
13:3191-3197 (1999)). Products are analyzed by electrophoresis in 15% or 18%
polyacrylamide sequencing gels using appropriate RNA standards. By monitoring
the gels
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for radioactivity, the natural production of 10 to 25 nt RNAs from the double
stranded
RNA can be determined.
The band of double stranded RNA, about 21-23 bps, is eluded. The efficacy of
these 21-23 mers for suppressing NOVX transcription is assayed in vitro using
the same
rabbit reticulocyte assay described above using 50 nanomolar of double
stranded 21-23 mer
for each assay. The sequence of these 21-23 mers is then determined using
standard
nucleic acid sequencing techniques.
RNA Preparation
21 nt RNAs, based on the sequence determined above, are chemically synthesized
using Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo,
Germany). Synthetic oligonucleotides are deprotected and gel-purified
(Elbashir,
Lendeckel, & Tuschl, Genes & Dev. 15, 188-200 (2001)), followed by Sep-Pak C18
cartridge (Waters, Milford, Mass., USA) purification (Tuschl, et al.,
Biochemistry,
32:11658-11668 (1993)).
These RNAs (20 p,M) single strands are incubated in annealing buffer (100 mM
potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1
min at
90° C followed by 1 h at 37° C.
CeII Culture
A cell culture known in the art to regularly express NOVX is propagated using
standard conditions. 24 hours before transfection, at approx. 80% confluency,
the cells are
trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3 X 105
cells/ml) and
transferred to 24-well plates (500 ml/well). Transfection is performed using a
commercially available lipofection kit and NOVX expression is monitored using
standard
techniques with positive and negative control. A positive control is cells
that naturally
express NOVX while a negative control is cells that do not express NOVX. Base-
paired 21
and 22 nt siRNAs with overhanging 3' ends mediate efficient sequence-specific
mRNA
degradation in lysates and in cell culture. Different concentrations of siRNAs
are used. An
efficient concentration for suppression in vitro in mammalian culture is
between 25 nM to
100 nM final concentration. This indicates that siRNAs are effective at
concentrations that
are several orders of magnitude below the concentrations applied in
conventional antisense
or ribozyme gene targeting experiments.



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The above method provides a way both for the deduction of NOVX siRNA
sequence and the use of such siRNA for in vitro suppression. In vivo
suppression may be
performed using the same siRNA using well known in vivo transfection or gene
therapy
transfection techniques.
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to the nucleic acid molecule
comprising the
nucleotide sequence of SEQ )D NO:2n-1, wherein n is an integer between l and
606, or
fragments, analogs or derivatives thereof. An "antisense" nucleic acid
comprises a
nucleotide sequence that is complementary to a "sense" nucleic acid encoding a
protein
(e.g., complementary to the coding strand of a double-stranded cDNA molecule
or
complementary to an mRNA sequence). In specific aspects, antisense nucleic
acid
molecules are provided that comprise a sequence complementary to at least
about 10, 25,
50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a
portion
thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and
analogs of
a NOVX protein of SEQ m N0:2n, wherein n is an integer between 1 and 606, or
antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ m
N0:2n-1, wherein n is an integer between 1 and 606, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence encoding a NOVX protein.
The term
"coding region" refers to the region of the nucleotide sequence comprising
codons which
are translated into amino acid residues. In another embodiment, the antisense
nucleic acid
molecule is antisense to a "noncoding region" of the coding strand of a
nucleotide sequence
encoding the NOVX protein. The term "noncoding region" refers to 5' and 3'
sequences
which flank the coding region that are not translated into amino acids (i.e.,
also referred to
as 5' and 3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein,
antisense nucleic acids of the invention can be designed according to the
rules of Watson
and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can
be
complementary to the entire coding region of NOVX mRNA, but more preferably is
an
oligonucleotide that is antisense to only a portion of the coding or noncoding
region of
NOVX mRNA. For example, the antisense oligonucleotide can be complementary to
the
region surrounding the translation start site of NOVX ml2NA. An antisense
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oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45
or 50
nucleotides in length. An antisense nucleic acid of the invention can be
constructed using
chemical synthesis or enzymatic ligation reactions using procedures known in
the art. For
example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically
synthesized using naturally-occurnng nucleotides or variously modified
nucleotides
designed to increase the biological stability of the molecules or to increase
the physical
stability of the duplex formed between the antisense and sense nucleic acids
(e.g.,
phosphorothioate derivatives and acridine substituted nucleotides can be
used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouridine,
5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil,
dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil,
4-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively,
the antisense nucleic acid can be produced biologically using an expression
vector into
which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed
from the inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of
interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated in situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding a NOVX protein to thereby inhibit expression of the
protein (e.g.,
by inhibiting transcription and/or translation). The hybridization can be by
conventional
nucleotide complementarity to form a stable duplex, or, for example, in the
case of an
antisense nucleic acid molecule that binds to DNA duplexes, through specific
interactions
in the major groove of the 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
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then administered systemically. For example, for systemic administration,
antisense
molecules can be modified such that they specifically bind to receptors or
antigens
expressed on a selected cell surface (e.g., by linking the antisense nucleic
acid molecules to
peptides or antibodies that bind to cell surface receptors or antigens). The
antisense nucleic
acid molecules can also be delivered to cells using the vectors described
herein. To achieve
sufficient nucleic acid molecules, vector constructs in which the antisense
nucleic acid
molecule is placed under the control of a strong pol II or pol III promoter
are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is
an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
(3-units,
the strands run parallel to each other. See, e.g., Gaultier, et al., 1987.
Nucl. Acids Res. 15:
6625-6641. The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (See, e.g., moue, et al. 1987. Nuel. Acids Res. 15:
6131-6148) or
a chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987. FEBSLett. 215: 327-
330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified
bases, and nucleic acids whose sugar phosphate backbones are modified or
derivatized.
These modifications are carned out at least in part to enhance the chemical
stability of the
modified nucleic acid, such that they may be used, for example, as antisense
binding
nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described
in
Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically
cleave
NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme
having specificity for a NOVX-encoding nucleic acid can be designed based upon
the
nucleotide sequence of a NOVX cDNA disclosed herein (i.e., SEQ ID N0:2n-1,
wherein n
is an integer between 1 and 606). For example, a derivative of a TetralZymena
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 NOVX-encoding
mRNA.
See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to
Cech, et al.
NOVX mRNA can also be used to select a catalytic RNA having a specific
ribonuclease
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activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993)
Science
261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the NOVX nucleic acid
(e.g., the
NOVX promoter and/or enhancers) to form triple helical structures that prevent
transcription of the NOVX gene in target cells. See, e.g., Helene, 1991.
Anticancer Drug
Des. 6: 569-84; Helene, et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher,
1992.
Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization,
or solubility of the molecule. For example, the deoxyribose phosphate backbone
of the
nucleic acids can be modified to generate peptide nucleic acids. See, e.g.,
Hyrup, et al.,
1996. BioorgMed Chem 4: 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
nucleotide bases 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 oligomer 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: 14670-14675. -
PNAs of NOVX can be used in therapeutic and diagnostic applications. For
example, PNAs can be used as antisense or antigene agents for sequence-
specific
modulation of gene expression by, e.g., inducing transcription or translation
arrest or
inhibiting replication. PNAs of NOVX can also be used, for example, in the
analysis of
single base pair mutations in a gene (e.g., PNA directed PCR clamping; as
artificial
restriction enzymes when used in combination with other enzymes, e.g., S1
nucleases (See,
Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and
hybridization
(See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of NOVX can be
generated
that may combine the advantageous properties of PNA and DNA. Such chimeras
allow
DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with
the DNA
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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 nucleotide bases, and orientation
(see, Hyrup,
et al., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as
described
in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-
3363. For
example, a DNA chain can be synthesized on a solid support using standard
phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used
between the
PNA and the 5' end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17:
5973-5988.
PNA monomers are then coupled in a stepwise manner to produce a chimeric
molecule
with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra.
Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and
a 3' PNA
segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-
11124.
In other embodiments, the oligonucleotide may include other appended groups
such
as peptides (e.g., for targeting host cell receptors ifa vivo), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger, et al., 1989. Pr~e. Natl.
Acad. Sci. U.SA. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
WO88/09810) 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. BioTechfaiques 6:958-976) or intercalating
agents (see, e.g.,
Zon, 1988. Pharrn. Res. 5: 539-549). To this end, the oligonucleotide may be
conjugated to
another molecule, e.g., a peptide, a hybridization triggered cross-linking
agent, a transport
agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino acid sequence of NOVX polypeptides whose sequences are provided in any
one of
SEQ III N0:2ra, wherein n is an integer between l and 606. The invention also
includes a
mutant or variant protein any of whose residues may be changed from the
corresponding
residues shown in any one of SEQ ID N0:2n, wherein n is an integer between 1
and 606,
while still encoding a protein that maintains its NOVX activities and
physiological
functions, or a functional fragment thereof.
In general, a NOVX variant that preserves NOVX-like function includes any
variant
in which residues at a particular position in the sequence have been
substituted by other



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amino acids, and further include the possibility of inserting an additional
residue or
residues between two residues of the parent protein as well as the possibility
of deleting
one or more residues from the parent sequence. Any amino acid substitution,
insertion, or
deletion is encompassed by the invention. In favorable circumstances, the
substitution is a
conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and
biologically-active portions thereof, or derivatives, fragments, analogs or
homologs thereof.
Also provided are polypeptide fragments suitable for use as immunogens to
raise
anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated
from
cells or tissue sources by an appropriate purification scheme using standard
protein
purification techniques. Tn another embodiment, NOVX proteins are produced by
recombinant DNA techniques. Alternative to recombinant expression, a NOVX
protein or
polypeptide can be synthesized chemically using standard peptide synthesis
techniques.
An "isolated" or "purified" polypeptide or 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 NOVX protein is derived, or substantially
free from
chemical precursors or other chemicals when chemically synthesized. The
language
"substantially free of cellular material" includes preparations of NOVX
proteins in which
the protein is separated from cellular components of the cells from which it
is isolated or
recombinantly-produced. In one embodiment, the language "substantially free of
cellular
material" includes preparations of NOVX proteins having less than about 30%
(by dry
weight) of non-NOVX proteins (also referred to herein as a "contaminating
protein"), more
preferably less than about 20% of non-NOVX proteins, still more preferably
less than about
10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX
proteins. When the NOVX protein or biologically-active portion thereof is
recombinantly-produced, it is also preferably substantially free of culture
medium, i.e.,
culture medium represents less than about 20%, more preferably less than about
10%, and
most preferably less than about 5% of the volume of the NOVX protein
preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from
chemical
precursors or other chemicals that are involved in the synthesis of the
protein. In one
embodiment, the language "substantially free of chemical precursors or other
chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry
weight) of
chemical precursors or non-NOVX chemicals, more preferably less than about 20%
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chemical precursors or non-NOVX chemicals, still more preferably less than
about 10%
chemical precursors or non-NOVX chemicals, and most preferably less than about
5%
chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequences of the
NOVX proteins (e.g., the amino acid sequence of SEQ ID NO:2n, wherein n is an
integer
between I and 606) that include fewer amino acids than the full-length NOVX
proteins,
and exhibit at least one activity of a NOVX protein. Typically, biologically-
active portions
comprise a domain or motif with at least one activity of the NOVX protein. A
biologically-active portion of a NOVX protein can be a polypeptide which is,
for example,
10, 25, 50, 100 or more amino acid residues in length.
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 NOVX protein.
1 S In an embodiment, the NOVX protein has an amino acid sequence of SEQ lD
N0:2n, wherein n is an integer between l and 606. In other embodiments, the
NOVX
protein is substantially homologous to SEQ ID N0:2n, wherein n is an integer
between 1
and 606, and retains the functional activity of the protein of SEQ 1D N0:2n,
wherein n is
an integer between 1 and 606, yet differs in amino acid sequence due to
natural allelic
variation or mutagenesis, as described in detail, below. Accordingly, in
another
embodiment, the NOVX protein is a protein that comprises an amino acid
sequence at least
about 45% homologous to the amino acid sequence of SEQ lD N0:2n, wherein n is
an
integer between l and 606, and retains the functional activity of the NOVX
proteins of
SEQ JD N0:2ra, wherein n is an integer between 1 and 606.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be
introduced in the sequence of a first amino acid or nucleic acid sequence for
optimal
alignment with a second amino 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
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molecules are homologous at that position (i.e., as used herein amino acid or
nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known in the art, such as GAP software provided in the GCG program package.
See,
Needleman and Wunsch, 1970. JMoI Biol 48: 443-453. Using GCG GAP software with
the following settings for nucleic acid sequence comparison: GAP creation
penalty of 5.0
and GAP extension penalty of 0.3, the coding region of the analogous nucleic
acid
sequences referred to above exhibits a degree of identity preferably of at
least 70%, 75%,
80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA
sequence
of SEQ ID N0:2n-1, wherein n is an integer between l and 606.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence. identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I,
in the case of
nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing
the number of matched positions by the total number of positions in the region
of
comparison (i.e., the window size), and multiplying the result by 100 to yield
the
percentage of sequence identity. The term "substantial identity" as used
herein denotes a
characteristic of a polynucleotide sequence, wherein the polynucleotide
comprises a
sequence that has at least 80 percent sequence identity, preferably at least
85 percent
identity and often 90 to 95 percent sequence identity, more usually at least
99 percent
sequence identity as compared to a reference sequence over a comparison
region.
Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein,
a
NOVX "chimeric protein" or "fusion protein" comprises a NOVX polypeptide
operatively-linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to
a
polypeptide having an amino acid sequence corresponding to a NOVX protein of
SEQ ID
N0:2fz, wherein n is an integer between 1 and 606, whereas a "non-NOVX
polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a
protein that is not
substantially homologous to the NOVX protein, e.g., a protein that is
different from the
NOVX protein and that is derived from the same or a different organism. Within
aNOVX
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fusion protein the NOVX polypeptide can correspond to all or a portion of a
NOVX
protein. In one embodiment, a NOVX fusion protein comprises at least one
biologically-active portion of a NOVX protein. In another embodiment, a NOVX
fusion
protein comprises at least two biologically-active portions of a NOVX protein.
In yet
another embodiment, a NOVX fusion protein comprises at least three
biologically-active
portions of a NOVX protein. Within the fusion protein, the term "operatively-
linked" is
intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide
are fused
in-frame with one another. The non-NOVX polypeptide can be fused to the N-
terminus or
C-terminus of the NOVX polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which
the
NOVX sequences are fused to the C-terminus of the GST (glutathione S-
transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
NOVX
polypeptides.
In another embodiment, the fusion protein is a NOVX protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host
cells), expression and/or secretion of NOVX can be increased through use of a
heterologous signal sequence.
In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion
protein in which the NOVX sequences are fused to sequences derived from a
member of
the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of
the
invention can be incorporated into pharmaceutical compositions and
administered to a
subject to inhibit an interaction between a NOVX ligand and a NOVX protein on
the
surface of a cell, to thereby suppress NOVX-mediated signal transduction in
vivo. The
NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability
of a
NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX 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. Moreover, the
NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens
to
produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in
screening
assays to identify molecules that inhibit the interaction of NOVX with a NOVX
ligand.
A NOVX chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g_, by employing blunt-ended or stagger-ended termini for
ligation, restriction
69



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enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as
appropriate, alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic
ligation. In another embodiment, the fusion gene can be synthesized by
conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of
gene fragments can be carried out using anchor primers that give rise to
complementary
overhangs between two consecutive gene fragments that can subsequently be
annealed and
reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al.
(eds.) CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many
expression vectors are commercially available that already encode a fusion
moiety (e.g., a
GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an
expression
vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists
The invention also pertains to variants of the NOVX proteins that function as
either
NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein
can be generated by mutagenesis (e.g., discrete point mutation or truncation
of the NOVX
protein). An agonist of the NOVX protein can retain substantially the same, or
a subset of,
the biological activities of the naturally occurring form of the NOVX protein.
An
antagonist of the NOVX protein can inhibit one or more of the activities of
the naturally
occurring form of the NOVX protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which includes
the NOVX
protein. Thus, specific biological effects can be elicited by treatment with a
variant of
limited function. In one embodiment, treatment of a subject with a variant
having a subset
of the biological activities of the naturally occurnng form of the protein has
fewer side
effects in a subject relative to treatment with the naturally occurring form
of the NOVX
proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i.e.,
mimetics) or as NOVX antagonists can be identified by screening combinatorial
libraries of
mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein
agonist or
antagonist activity. In one embodiment, a variegated library of NOVX variants
is
generated by combinatorial mutagenesis at the nucleic acid level and is
encoded by a
variegated gene library. A variegated library of NOVX variants can be produced
by, for
example, enzymatically ligating a mixture of synthetic oligonucleotides into
gene
sequences such that a degenerate set of potential NOVX sequences is
expressible as



CA 02488547 2004-12-02
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individual polypeptides, or alternatively, as a set of larger fusion proteins
(e.g., for phage
display) containing the set of NOVX sequences therein. There are a variety of
methods
which can be used to produce libraries of potential NOVX 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 of potential
NOVX sequences.
Methods for synthesizing degenerate oligonucleotides are well-known within the
art. See,
e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Aranu. Rev.
Biochem. 53: 323;
Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res.
11: 477.
Polypeptide Libraries
In addition, libraries of fragments of the NOVX protein coding sequences can
be
used to generate a variegated population of NOVX fragments for screening and
subsequent
selection of variants of a NOVX protein. In one embodiment, a library of
coding sequence
fragments can be generated by treating a double stranded PCR fragment of a
NOVX 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 that can include sense/antisense pairs from different
nicked products,
removing single stranded portions from reformed duplexes by treatment with SI
nuclease,
and ligating the resulting fragment library into an expression vector. By this
method,
expression libraries can be derived which encodes N-terminal and internal
fragments of
various sizes of the NOVX proteins.
Various 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 are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of NOVX
proteins. The most widely used techniques, which are amenable to high
throughput
analysis, for screening large gene libraries typically include cloning the
gene library into
repIicable expression vectors, transforming appropriate cells with the
resulting library of
vectors, and expressing the combinatorial genes under conditions in which
detection of a
desired activity facilitates isolation of the vector encoding the gene whose
product was
detected. Recursive ensemble mutagenesis (REM), a new technique that enhances
the
frequency of functional mutants in the libraries, can be used in combination
with the
71



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screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan,
1992. Proc.
Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein
Engineering
6:327-331.
Anti-NOVX Antibodies
Included in the invention are antibodies to NOVX proteins, or fragments of
NOVX
proteins. The term "antibody" as used herein refers to immunoglobulin
molecules and
immunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that
contain an antigen binding site that specifically binds (immunoreacts with) an
antigen.
Such antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, single
chain, Fab, Fab' and F~ab~~2 fragments, and an Fab expression library. In
general, antibody
molecules obtained from humans relates to any of the classes IgG, IgM, IgA,
IgE and IgD,
which differ from one another by the nature of the heavy chain present in the
molecule.
Certain classes have subclasses as well, such as IgGI, IgG2, and others.
Furthermore, in
humans, the light chain may be a kappa chain or a lambda chain. Reference
herein to
antibodies includes a reference to all such classes, subclasses and types of
human antibody
species.
An isolated protein of the invention intended to serve as an antigen, or a
portion or
fragment thereof, can be used as an immunogen to generate antibodies that
immunospecifically bind the antigen, using standard techniques for polyclonal
and
monoclonal antibody preparation. The full-length protein can be used or,
alternatively, the
invention provides antigenic peptide fragments of the antigen for use as
immunogens. An
antigenic peptide fragment comprises at least 6 amino acid residues of the
amino acid
sequence of the full length protein, such as an amino acid sequence of SEQ ID
N0:2n,
wherein n is an integer between 1 and 606, and encompasses an epitope thereof
such that
an antibody raised against the peptide forms a specific immune complex with
the full
length protein or with any fragment that contains the epitope. Preferably, the
antigenic
peptide comprises at least 10 amino acid residues, or at least 15 amino acid
residues, or at
least 20 amino acid residues, or at least 30 amino acid residues. Preferred
epitopes
encompassed by the antigenic peptide are regions of the protein that are
located on its
surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of NOVX that is located on the surface of the
protein, e.g., a
hydrophilic region. A hydrophobicity analysis of the human NOVX protein
sequence will
72



CA 02488547 2004-12-02
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indicate which regions of a NOVX polypeptide are particularly hydrophilic and,
therefore,
are likely to encode surface residues useful for targeting antibody
production. As a means
for targeting antibody production, hydropathy plots showing regions of
hydrophilicity and
hydrophobicity may be generated by any method well known in the art,
including, for
example, the Kyte Doolittle or the Hopp Woods methods, either with or without
Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each
incorporated herein
by reference in their entirety. Antibodies that are specific for one or more
domains within
an antigenic protein, or derivatives, fragments, analogs or homologs thereof,
are also
provided herein.
The term "epitope" includes any protein determinant capable of specific
binding to
an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of
chemically
active surface groupings of molecules such as amino acids or sugar side chains
and usually
have specific three dimensional structural characteristics, as well as
specific charge
characteristics. A NOVX polypeptide or a fragment thereof comprises at least
one antigenic
epitope. An anti-NOVX antibody of the present invention is said to
specifically bind to
antigen NOVX when the equilibrium binding constant (KD) is _<1 ~,M, preferably
<_ 100
nM, more preferably <_ 10 nM, and most preferably <_ 100 pM to about 1 pM, as
measured
by assays including radioligand binding assays or similar assays known to
skilled artisans.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of
polyclonal or monoclonal antibodies directed against a protein of the
invention, or against
derivatives, fragments, analogs homologs or orthologs thereof (see, for
example,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).
Some of
these antibodies are discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals
(e.g.,
rabbit, goat, mouse or other mammal) may be immunized by one or more
injections with
the native protein, a synthetic variant thereof, or a derivative of the
foregoing. An
appropriate immunogenic preparation can contain, for example, the naturally
occurring
73



CA 02488547 2004-12-02
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immunogenic protein, a chemically synthesized polypeptide representing the
immunogenic
protein, or a recombinantly expressed immunogenic protein. Furthermore, the
protein may
be conjugated to a second protein known to be immunogenic in the mammal being
immunized. Examples of such immunogenic proteins include but are not limited
to
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin
inhibitor. The preparation can further include an adjuvant. Various adjuvants
used to
increase the immunological response include, but are not limited to, Freund's
(complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface active
substances (e.g.,
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium
paavum,
or similar immunostimulatory agents. Additional examples of adjuvants which
can be
employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
1 S isolated from the mammal (e.g., from the blood) and further purified by
well known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the immunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for example, by
D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA,
Vol. 14, No. 8
(April 17, 2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs)
of the monoclonal antibody are identical in all the molecules of the
population. MAbs thus
contain an antigen binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by I~ohler and Milstein, Nature, 256:495 (1975). In a hybridoma
method, a
mouse, hamster, or other appropriate host animal, is typically immunized with
an
74



CA 02488547 2004-12-02
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immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies
that will specifically bind to the immunizing agent. Alternatively, the
lymphocytes can be
immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof
or a fusion protein thereof. Generally, either peripheral blood lymphocytes
are used if cells
of human origin are desired, or spleen cells or lymph node cells are used if
non-human
mammalian sources are desired. The lymphocytes are then fused with an
immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian cells,
particularly
myeloma cells of rodent, bovine and human origin. Usually, rat or mouse
myeloma cell
lines are employed. The hybridoma cells can be cultured in a suitable culture
medium that
preferably contains one or more substances that inhibit the growth or survival
of the
unfused, immortalized cells. For example, if the parental cells lack the
enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium
for the hybridomas typically will include hypoxanthine, aminopterin, and
thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell lines are murine
myeloma lines, which can be obtained, for instance, from the Salk Institute
Cell
Distribution Center, San Diego, California and the American Type Culture
Collection,
Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines
also
have been described for the production of human monoclonal antibodies (Kozbor,
J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques
and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be
assayed
for the presence of monoclonal antibodies directed against the antigen.
Preferably, the
binding specificity of monoclonal antibodies produced by the hybridoma cells
is
determined by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such
techniques and assays are known in the art. The binding affinity of the
monoclonal
antibody can, for example, be determined by the Scatchard analysis of Munson
and Pollard,
Anal. Biochem., 107:220 (1980). It is an objective, especially important in
therapeutic



CA 02488547 2004-12-02
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applications of monoclonal antibodies, to identify antibodies having a high
degree of
specificity and a high binding affinity for the target antigen.
After the desired hybridoma cells are identified, the clones can be subcloned
by
limiting dilution procedures and grown by standard methods (Goding,1986).
Suitable
culture media for this purpose include, for example, Dulbecco's Modified
Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo
as
ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified
from the culture medium or ascites fluid by conventional immunoglobulin
purification
procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography,
gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such
as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies
of the invention can be readily isolated and sequenced using conventional
procedures (e.g.,
by using oligonucleotide probes that are capable of binding specifically to
genes encoding
the heavy and light chains of marine antibodies). The hybridoma cells of the
invention
serve as a preferred source of such DNA. Once isolated, the DNA can be placed
into
expression vectors, which are then transfected into host cells such as simian
COS cells,
Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce
immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in
the
recombinant host cells. The DNA also can be modified, for example, by
substituting the
coding sequence for human heavy and light chain constant domains in place of
the
homologous marine sequences (U.S. Patent No. 4,816,567; Mornson, Nature 368,
812-13
(1994)) or by covalently joining to the immunoglobulin coding sequence all or
part of the
coding sequence for a non-immunoglobulin polypeptide. Such a non-
immunoglobulin
polypeptide can be substituted for the constant domains of an antibody of the
invention, or
can be substituted for the variable domains of one antigen-combining site of
an antibody of
the invention to create a chimeric bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against
the administered immunoglobulin. Humanized forms of antibodies are chimeric
76



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immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
F(ab')2 or other antigen-binding subsequences of antibodies) that are
principally comprised
of the sequence of a human immunoglobulin, and contain minimal sequence
derived from a
non-human immunoglobulin. Humanization can be performed following the method
of
Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature,
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting
rodent CDRs or CDR sequences for the corresponding sequences of a human
antibody.
(See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues
of the
human immunoglobulin are replaced by corresponding non-human residues.
Humanized
antibodies can also comprise residues which are found neither in the recipient
antibody nor
in the imported CDR or framework sequences. In general, the humanized antibody
will
comprise substantially all of at least one, and typically two, variable
domains, in which all
or substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the framework regions are those
of a human
immunoglobulin consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta,
Curr. Op.
Struct. Biol., 2:593-596 (1992)).
Human Antibodies
Fully human antibodies essentially relate to antibody molecules in which the
entire
sequence of both the light chain and the heavy chain, including the CDRs,
arise from
human genes. Such antibodies are termed "human antibodies", or "fully human
antibodies"
herein. Human monoclonal antibodies can be prepared by the trioma technique;
the human
B-cell hybridoma technique (see I~ozbor, et al., 1983 Immunol Today 4: 72) and
the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
Human
monoclonal antibodies may be utilized in the practice of the present invention
and may be
produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci
USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro
(see Cole, et
al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,
pp.
77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991);
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CA 02488547 2004-12-02
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Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in all respects, including gene rearrangement, assembly, and antibody
repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technology 10,
779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Mornson ( Nature
368,
812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996));
Neuberger
(Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol.
13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals which are modified so as to produce fully human antibodies rather than
the
animal's endogenous antibodies in response to challenge by an antigen. (See
PCT
publication W094/02602). The endogenous genes encoding the heavy and light
immunoglobulin chains in the nonhuman host have been incapacitated, and active
loci
encoding human heavy and light chain immunoglobulins are inserted into the
host's
genome. The human genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal which
provides
all the desired modifications is then obtained as progeny by crossbreeding
intermediate
transgenic animals containing fewer than the full complement of the
modifications. The
preferred embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse~ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This
animal produces B cells which secrete fully human immunoglobulins. The
antibodies can
be obtained directly from the animal after immunization with an immunogen of
interest, as,
for example, a preparation of a polyclonal antibody, or alternatively from
immortalized B
cells derived from the animal, such as hybridomas producing monoclonal
antibodies.
Additionally, the genes encoding the immunoglobulins with human variable
regions can be
recovered and expressed to obtain the antibodies directly, or can be further
modified to
obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S.
Patent No. 5,939,598. It can be obtained by a method including deleting the J
segment
genes from at least one endogenous heavy chain locus in an embryonic stem cell
to prevent
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rearrangement of the locus and to prevent formation of a transcript of a
rearranged
immunoglobulin heavy chain locus, the deletion being effected by a targeting
vector
containing a gene encoding a selectable marker; and producing from the
embryonic stem
cell a transgenic mouse whose somatic and germ cells contain the gene encoding
the
selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression
vector that
contains a nucleotide sequence encoding a heavy chain into one mammalian host
cell in
culture, introducing an expression vector containing a nucleotide sequence
encoding a light
chain into another mammalian host cell, and fusing the two cells to form a
hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and the light
chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an antigenic protein of the invention (see
e.g., U.S.
Patent No. 4,946,778). In addition, methods can be adapted for the
construction of Fab
expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to
allow rapid and
effective identification of monoclonal Fab fragments with the desired
specificity for a
protein or derivatives, fragments, analogs or homologs thereof. Antibody
fragments that
contain the idiotypes to a protein antigen may be produced by techniques known
in the art
including, but not limited to: (i) an F(ab~)a fragment produced by pepsin
digestion of an
antibody molecule; (ii) an Fab fragment generated by reducing the disulfide
bridges of an
F(ab~)a fragment; (iii) an Fab fragment generated by the treatment of the
antibody molecule
with papain and a reducing agent and (iv) F~ fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies
that have binding specificities for at least two different antigens. In the
present case, one of
the binding specificities is for an antigenic protein of the invention. The
second binding
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target is any other antigen, and advantageously is a cell-surface protein or
receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas)
produce a potential mixture of ten different antibody molecules, of which only
one has the
correct bispecific structure. The purification of the correct molecule is
usually
accomplished by affinity chromatography steps. Similar procedures are
disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-
3659
(1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least
part of the hinge, CH2, and CH3 regions. It is preferred to have the first
heavy-chain
constant region (CH1) containing the site necessary for light-chain binding
present in at
least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if
desired, the immunoglobulin light chain, are inserted into separate expression
vectors, and
are co-transfected into a suitable host organism. For further details of
generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210
(1986).
According to another approach described in WO 96/27011, the interface between
a
pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
which are recovered from recombinant cell culture. The preferred interface
comprises at
least a part of the CH3 region of an antibody constant domain. In this method,
one or more
small amino acid side chains from the interface of the first antibody molecule
are replaced
with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical
or similar size to the large side chains) are created on the interface of the
second antibody
molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or
threonine). This provides a mechanism for increasing the yield of the
heterodimer over
other unwanted end-products such as homodimers.
Bispecif c antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating
bispecific
antibodies from antibody fragments have been described in the literature. For
example,



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bispecific antibodies can be prepared using chemical linkage. Brennan et al.,
Science
229:81 (1985) describe a procedure wherein intact antibodies are
proteolytically cleaved to
generate F(ab')2 fragments. These fragments are reduced in the presence of the
dithiol
complexing agent sodium arsenite to stabilize vicinal dithiols and prevent
intermolecular
S disulfide formation. The Fab' fragments generated are then converted to
thionitrobenzoate
(TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the
Fab'-thiol
by reduction with mercaptoethylamine and is mixed with an equimolar amount of
the other
Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies
produced
can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')Z
molecule. Each
Fab' fragment was separately secreted from E. coli and subjected to directed
chemical
coupling in vitro to form the bispecific antibody. The bispecific antibody
thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well
as trigger the lytic activity of human cytotoxic lymphocytes against human
breast tumor
targets.
Various techniques for making and isolating bispecific antibody fragments
directly
from recombinant cell culture have also been described. For example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553
(1992). The leucine zipper peptides from the Fos and Jun proteins were linked
to the Fab'
portions of two different antibodies by gene fusion. The antibody homodimers
were
reduced at the hinge region to form monomers and then re-oxidized to form the
antibody
heterodimers. This method can also be utilized for the production of antibody
homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
USA
90:6444-6448 (1993) has provided an alternative mechanism for making
bispecific
antibody fragments. The fragments comprise a heavy-chain variable domain (VH)
connected to a light-chain variable domain (VL) by a linker which is too short
to allow
pairing between the two domains on the same chain. Accordingly, the VH and VL
domains
of one fragment are forced to pair with the complementary VL and VH domains of
another
fragment, thereby forming two antigen-binding sites. Another strategy for
making
bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has
also been
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
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Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60
(1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of
which originates in the protein antigen of the invention. Alternatively, an
anti-antigenic
arm of an immunoglobulin molecule can be combined with an arm which binds to a
triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.
CD2, CD3,
CD28, or B7), or Fc receptors for IgG (Fc~yR), such as FcyRI (CD64), Fc~yRII
(CD32) and
Fc~yRIII (CD16) so as to focus cellular defense mechanisms to the cell
expressing the
particular antigen. Bispecific antibodies can also be used to direct cytotoxic
agents to cells
which express a particular antigen. These antibodies possess an antigen-
binding arm and
an arm which binds a cytotoxic agent or a radionuclide chelator, such as
EOTUBE, DPTA,
DOTA, or TETA. Another bispecific antibody of interest binds the protein
antigen
described herein and further binds tissue factor (TF)
Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such
antibodies have, for example, been proposed to target immune system cells to
unwanted
cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared
in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking
agents. For example, immunotoxins can be constructed using a disulfide
exchange reaction
or by forming a thioether bond. Examples of suitable reagents for this purpose
include
iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for
example, in U.S.
Patent No. 4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing
interchain disulfide bond formation in this region. The homodimeric antibody
thus
generated can have improved internalization capability and/or increased
complement-mediated cell killing and antibody-dependent cellular cytotoxicity
(ADCC}.
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol.,
148:
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2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can
also be
prepared using heterobifunctional cross-linkers as described in Wolff et al.
Cancer
Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered
that has
dual Fc regions and can thereby have enhanced complement lysis and ADCC
capabilities.
See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g.,
an
enzymatically active toxin of bacterial, fungal, plant, or animal origin, or
fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above. Enzymatically active toxins and fragments thereof that
can be used
include diphtheria A chain, nonbinding active fragments of diphtheria toxin,
exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A
chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca
americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,
enomycin, and the
tricothecenes. A variety of radionuclides are available for the production of
radioconjugated antibodies. Examples include aI2Bi, Isila ~3y~ 9ola ~d is6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling agents such as N-succinimidyl-3-(2-
pyridyldithiol)
propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as
dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate),
aldehydes
(such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene
2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be
prepared as
described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA)
is an
exemplary chelating agent for conjugation of radionucleotide to the antibody.
See
W094/11026.
83



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In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate
is administered to the patient, followed by removal of unbound conjugate from
the
circulation using a clearing agent and then administration of a "ligand"
(e.g., avidin) that is
in turn conjugated to a cytotoxic agent.
Immunoliposomes
The antibodies disclosed herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art,
such as
described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985);
Hwang et al.,
Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and
4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation
method with a lipid composition comprising phosphatidylcholine, cholesterol,
and
PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded
through
filters of defined pore size to yield liposomes with the desired diameter.
Fab' fragments of
the antibody of the present invention can be conjugated to the liposomes as
described in
Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-
interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within
the liposome.
See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
Diagnostic Applications of Antibodies Directed Against the Proteins of the
Invention
In one embodiment, methods for the screening of antibodies that possess the
desired
specificity include, but are not limited to, enzyme linked immunosorbent assay
(ELISA)
and other immunologically mediated techniques known within the art. In a
specific
embodiment, selection of antibodies that are specific to a particular domain
of an NOVX
protein is facilitated by generation of hybridomas that bind to the fragment
of an NOVX
protein possessing such a domain. Thus, antibodies that are specific for a
desired domain
within an NOVX protein, or derivatives, fragments, analogs or homologs
thereof, are also
provided herein.
Antibodies directed against a NOVX protein of the invention may be used in
methods known within the art relating to the localization and/or quantitation
of a NOVX
protein (e.g., for use in measuring levels of the NOVX protein within
appropriate
physiological samples, for use in diagnostic methods, for use in imaging the
protein, and
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the like). In a given embodiment, antibodies specific to a NOVX protein, or
derivative,
fragment, analog or homolog thereof, that contain the antibody derived antigen
binding
domain, are utilized as pharmacologically active compounds (referred to
hereinafter as
"Therapeutics").
An antibody specific for a NOVX protein of the invention (e.g., a monoclonal
antibody or a polyclonal antibody) can be used to isolate a NOVX polypeptide
by standard
techniques, such as immunoaffinity, chromatography or immunoprecipitation. An
antibody
to a NOVX polypeptide can facilitate the purification of a natural NOVX
antigen from
cells, or of a recombinantly produced NOVX antigen expressed in host cells.
Moreover,
such an anti-NOVX antibody can be used to detect the antigenic NOVX protein
(e.g., in a
cellular lysate or cell supernatant) in order to evaluate the abundance and
pattern of
expression of the antigenic NOVX protein. Antibodies directed against a NOVX
protein
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 (i.e., physically linking) 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, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of
bioluminescent materials include luciferase, luciferin, and aequorin, and
examples of
suitable radioactive material include ~ZSI, 131h ssS or 3H.
Antibody Therapeutics
Antibodies of the invention, including polyclonal, monoclonal, humanized and
fully
human antibodies, may used as therapeutic agents. Such agents will generally
be employed
to treat or prevent a disease or pathology in a subject. An antibody
preparation, preferably
one having high specificity and high affinity for its target antigen, is
administered to the
subj ect and will generally have an effect due to its binding with the target.
Such an effect
may be one of two kinds, depending on the specific nature of the interaction
between the
given antibody molecule and the target antigen in question. In the first
instance,



CA 02488547 2004-12-02
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administration of the antibody may abrogate or inhibit the binding of the
target with an
endogenous ligand to which it naturally binds. In this case, the antibody
binds to the target
and masks a binding site of the naturally occurring ligand, wherein the ligand
serves as an
effector molecule. Thus the receptor mediates a signal transduction pathway
for which
ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a
physiological
result by virtue of binding to an effector binding site on the target
molecule. In this case
the target, a receptor having an endogenous ligand which may be absent or
defective in the
disease or pathology, binds the antibody as a surrogate effector ligand,
initiating a
receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates
generally
to the amount needed to achieve a therapeutic objective. As noted above, this
may be a
binding interaction between the antibody and its target antigen that, in
certain cases,
interferes with the functioning of the target, and in other cases, promotes a
physiological
response. The amount required to be administered will furthermore depend on
the binding
affinity of the antibody for its specific antigen, and will also depend on the
rate at which an
administered antibody is depleted from the free volume other subject to which
it is
administered. Common ranges for therapeutically effective dosing of an
antibody or
antibody fragment of the invention may be, by way of nonlimiting example, from
about 0.1
mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may
range, for example, from twice daily to once a week.
Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a protein of the invention, as well as other
molecules identified by the screening assays disclosed herein, can be
administered for the
treatment of various disorders in the form of pharmaceutical compositions.
Principles and
considerations involved in preparing such compositions, as well as guidance in
the choice
of components are provided, for example, in Remington : The Science And
Practice Of
Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton,
Pa. : 1995;
Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And
Trends,
Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein
Drug
Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
If the antigenic protein is intracellular and whole antibodies are used as
inhibitors,
internalizing antibodies are preferred. However, liposomes can also be used to
deliver the
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CA 02488547 2004-12-02
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antibody, or an antibody fragment, into cells. Where antibody fragments are
used, the
smallest inhibitory fragment that specifically binds to the binding domain of
the target
protein is preferred. For example, based upon the variable-region sequences of
an
antibody, peptide molecules can be designed that retain the ability to bind
the target protein
sequence. Such peptides can be synthesized chemically and/or produced by
recombinant
DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:
7889-7893
(1993). The formulation herein can also contain more than one active compound
as
necessary for the particular indication being treated, preferably those with
complementary
activities that do not adversely affect each other. Alternatively, or in
addition, the
composition can comprise an agent that enhances its function, such as, for
example, a
cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
Such
molecules are suitably present in combination in amounts that are effective
for the purpose
intended.
The active ingredients can also be entrapped in microcapsules prepared, for
example, by coacervation techniques or by interfacial polymerization, for
example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methyhnethacrylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes,
albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in
macroemulsions.
The formulations to be used for in vivo administration must be sterile. This
is
readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of solid
hydrophobic
polymers containing the antibody, which matrices are in the form of shaped
articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices include
polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y
ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic
acid copolymers such as the LUPRON DEPOT ~ (injectable microspheres composed
of
lactic acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl
acetate and
lactic acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels
release proteins for shorter time periods.
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ELISA Assay
An agent for detecting an analyte protein is an antibody capable of binding to
an
analyte 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 F(ab~~) can be used. The term "labeled", with regard to the
probe or antibody, is
intended to encompass direct labeling of the 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. Included within the usage of the term
"biological
sample", therefore, is blood and a fraction or component of blood including
blood serum,
blood plasma, or lymph. That is, the detection method of the invention can be
used to
detect an analyte mRNA, protein, or genomic DNA in a biological sample ira
vitro as well
as irz vivo. For example, in vitro techniques for detection of an analyte mRNA
include
Northern hybridizations and in situ hybridizations. In vitro techniques for
detection of an
analyte protein include enzyme linked immunosorbent assays (ELISAs), Western
blots,
immunoprecipitations, and immunofluorescence. In vitro techniques for
detection of an
analyte genomic DNA include Southern hybridizations. Procedures for conducting
immunoassays are described, for example in "ELISA: Theory and Practice:
Methods in
Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ,
1995;
"Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San
Diego,
CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen,
Elsevier
Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for
detection of an
analyte protein include introducing into a subject a labeled anti-an analyte
protein 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.
NOVX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding a NOVX protein, or derivatives, fragments,
analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule
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capable of transporting another nucleic acid to which it has been linked. One
type of vector
is a "plasmid", which refers to a circular double stranded DNA loop into which
additional
DNA segments can be ligated. Another type of vector is a viral vector, wherein
additional
DNA segments can be ligated into the viral genome. Certain vectors are capable
of
autonomous replication in a host cell into which they are introduced (e.g.,
bacterial vectors
having a bacterial origin of replication and episomal mammalian vectors).
Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the genome of a
host cell upon
introduction into the host cell, and thereby are replicated along with the
host genome.
Moreover, certain vectors are capable of directing the expression of genes to
which they are
operatively-linked. Such vectors are referred to herein as "expression
vectors". In general,
expression vectors of utility in recombinant DNA techniques are often in the
form of
plasmids. In the present specification, "plasmid" and "vector" can be used
interchangeably
as the plasmid is the most commonly used form of vector. However, the
invention is
intended to include such other forms of expression vectors, such as viral
vectors (e.g.,
replication defective retroviruses, adenoviruses and adeno-associated
viruses), which serve
equivalent functions.
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 means
that the recombinant expression vectors include one or more regulatory
sequences, selected
on the basis of the host cells to be used for expression, that is operatively-
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 sequences) in a manner that allows for expression of the
nucleotide
sequence (e.g., in an in vitro transcription/translation system or in a host
cell when the
vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers
and
other expression control elements (e.g., polyadenylation signals). Such
regulatory
sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:
METHODS 1N ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory
sequences include those that direct constitutive expression of a nucleotide
sequence in
many types of host cell and those that direct expression of the nucleotide
sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). 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,
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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., NOVX proteins, mutant forms of NOVX proteins,
fusion
proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression
of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX
proteins can be
expressed in bacterial cells such as Escherichia 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 ENZYMOLOGY 185, Academic
Press, San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be
transcribed and translated in vitro, for example using T7 promoter regulatory
sequences
and T7 polymerase.
Expression of proteins in prokaryotes is most often carned out in Escherichia
coli
with vectors containing constitutive or inducible 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 three purposes: (i) to increase expression of
recombinant
protein; (ii) to increase the solubility of the recombinant protein; and (iii)
to aid in the
purification of the recombinant protein by acting as a ligand in affinity
purification. Often,
in fusion expression vectors, a proteolytic cleavage site is introduced at the
junction of the
fusion moiety and the 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, Mass.) and
pRITS (Pharmacia, Piscataway, N.J.) that 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
(Amrann et al., (1988) Gene 69:301-315) and pET 1 ld (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990)
60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the
recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
1N



CA 02488547 2004-12-02
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ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another
strategy is
to alter the nucleic acid sequence of the nucleic acid to be inserted into an
expression vector
so that the individual codons for each amino acid are those preferentially
utilized in E. coli
(see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2I 18). Such
alteration of nucleic
S acid sequences of the invention can be carned out by standard DNA synthesis
techniques.
In another embodiment, the NOVX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Sacclaaromyces cerivisae include
pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,
Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells
(e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell.
Biol. 3: 2156-2165)
and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
I S In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC
(Kaufrnan,
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 expression systems for both prokaryotic and eukaryotic
cells see,
e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY
MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y., 1989.
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. Immuraol.
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. Acaa'. Sci. USA 86: 5473-5477),
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CA 02488547 2004-12-02
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pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; IJ.S. Pat. No. 4,873,316
and European
Application Publication No. 264,166). Developmentally-regulated promoters are
also
encompassed, e.g., the marine hox promoters (Kessel and Grass, 1990. Science
249:
374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev.
3:
537-546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation.
That is, the DNA molecule is operatively-linked to a regulatory sequence in a
manner that
I O allows for expression (by transcription of the DNA molecule) of an RNA
molecule that is
antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic
acid
cloned in the antisense orientation can be chosen that direct the continuous
expression of
the antisense RNA molecule in a variety of cell types, for instance viral
promoters and/or
enhancers, or regulatory sequences can be chosen that direct constitutive,
tissue specific or
cell type specific expression of antisense RNA. The antisense expression
vector can be in
the form of a recombinant plasmid, phagemid or attenuated virus in which
antisense nucleic
acids are produced under the control of a high efficiency regulatory region,
the activity of
which can be determined by the cell type into which the vector is introduced.
For a
discussion of the regulation of gene expression using antisense genes see,
e.g., Weintraub,
et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-
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 introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms
refer not only to the particular subject cell but also to the progeny or
potential progeny of
such a cell. Because certain modifications may occur in succeeding generations
due to
either mutation or environmental influences, such progeny may not, in fact, be
identical to
the parent cell, but are still included within the scope of the term as used
herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX
protein
can be expressed in bacterial cells such as E. coli, 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 in the art.
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Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting host cells
can be found
in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these '
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Various selectable
markers include those that confer resistance to drugs, such as 6418,
hygromycin and
methotrexate. Nucleic acid encoding a selectable marker can be introduced into
a host cell
on the same vector as that encoding NOVX or can be introduced on a separate
vector.
Cells stably transfected with the introduced nucleic acid can be identified by
drug selection
(e.g., cells that have incorporated the selectable marker gene will survive,
while the other
cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture,
can be used to produce (i.e., express) NOVX protein. Accordingly, the
invention further
provides methods for producing NOVX protein using the host cells of the
invention. In one
embodiment, the method comprises culturing the host cell of invention (into
which a
recombinant expression vector encoding NOVX protein has been introduced) in a
suitable
medium such that NOVX protein is produced. In another embodiment, the method
further
comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte
or an embryonic stem cell into which NOVX protein-coding sequences have been
introduced. Such host cells can then be used to create non-human transgenic
animals in
which exogenous NOVX sequences have been introduced into their genome or
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homologous recombinant animals in which endogenous NOVX sequences have been
altered. Such animals are useful for studying the function and/or activity of
NOVX protein
and for identifying and/or evaluating modulators of NOVX protein activity. As
used
herein, a "transgenic animal" is a non-human animal, preferably a mammal, more
preferably a rodent such as a rat or mouse, in which one or more of the cells
of the animal
includes a transgene. Other examples of transgenic animals include non-human
primates,
sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous
DNA that is
integrated into the genome of a cell from which a transgenic animal develops
and that
remains in the genome of the mature animal, thereby directing the expression
of an
encoded gene product in one or more cell types or tissues of the transgenic
animal. As used
herein, a "homologous recombinant animal" is a non-human animal, preferably a
mammal,
more preferably a mouse, in which an endogenous NOVX 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
NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte
(e.g., by
microinjection, retroviral infection) and allowing the oocyte to develop in a
pseudopregnant
female foster animal. The human NOVX cDNA sequences, i.e., any one of SEQ m
N0:2n-1, wherein n is an integer between 1 and 606, can be introduced as a
transgene into
the genome of a non-human animal. Alternatively, a non-human homologue of the
human
NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization
to the
human NOVX cDNA (described further supra) and used as a transgene. 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 NOVX transgene to direct expression of NOVX protein to
particular
cells. Methods for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become conventional in
the art and
are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and
4,873,191; and
Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. Similar methods are used for production of
other
transgenic animals. A transgenic founder animal can be identified based upon
the presence
of the NOVX transgene in its genome andlor expression of NOVX mRNA in tissues
or
cells of the animals. A transgenic founder animal can then be used to breed
additional
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animals carrying the transgene. Moreover, transgenic animals carrying a
transgene-encoding NOVX protein can further be bred to other transgenic
animals carrying
other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at
least a portion of a NOVX gene into which a deletion, addition or substitution
has been
introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The
NOVX gene
can be a human gene (e.g., the cDNA of any one of SEQ >D N0:2n-1, wherein n is
an
integer between 1 and 606), but more preferably, is a non-human homologue of a
human
NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID
NO:2n-l, wherein n is an integer between 1 and 606, can be used to construct a
homologous recombination vector suitable for altering an endogenous NOVX gene
in the
mouse genome. In one embodiment, the vector is designed such that, upon
homologous
recombination, the endogenous NOVX gene is functionally disrupted (i.e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination, the endogenous NOVX 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 NOVX protein). In the homologous
recombination
vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-
termini by
additional nucleic acid of the NOVX gene to allow for homologous recombination
to occur
between the exogenous NOVX gene carned by the vector and an endogenous NOVX
gene
in an embryonic stem cell. The additional flanking NOVX 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'-termini) are
included in the
vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of
homologous
recombination vectors. The vector is ten introduced into an embryonic stem
cell line (e.g.,
by electroporation) and cells in which the introduced NOVX gene has
homologously-recombined with the endogenous NOVX gene are selected. See, e.g.,
Li, et
al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to
form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152. A chimeric embryo can then be implanted into a suitable
pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the



CA 02488547 2004-12-02
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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. Curr.
S OpifZ. Biotechraol. 2:. 823-829; PCT International Publication Nos.: WO
90/11354; WO
91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain selected systems that allow for regulated expression of the transgene.
One example
of such a system is the cre/loxP recombinase system of bacteriophage 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. See, O'Gorman, et al., 1991. Science
251:1351-1355.
If a cre/loxP recombinase system is used to regulate expression of the
transgene, animals
containing transgenes encoding both the Cre recombinase and a selected protein
are
required. Such animals can be provided through the construction of "double"
transgenic
animals, e.g., by mating two transgenic animals, one containing a transgene
encoding a
selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilmut, et al., 1997. Nature 385: 810-
813. In brief,
a cell (e.g., a somatic cell) from the transgenic animal can be isolated and
induced to exit
the growth cycle and enter Go phase. The quiescent cell can then be fused,
e.g., through the
use of electrical pulses, to an enucleated oocyte from an animal of the same
species from
which the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it
develops to morula or blastocyte and then transferred to pseudopregnant female
foster
animal. The offspring borne of this female foster animal will be a clone of
the animal from
which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies
(also referred to herein as "active compounds") of the invention, and
derivatives, fragments,
analogs and homologs thereof, can be incorporated into pharmaceutical
compositions
suitable for administration. Such compositions typically comprise the nucleic
acid
molecule, protein, or antibody and a pharmaceutically acceptable carrier. As
used herein,
"pharmaceutically acceptable Garner" is intended to include any and all
solvents, dispersion
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media, coatings, antibacterial and antifungal agents, isotonic and absorption
delaying
agents, and the like, compatible with pharmaceutical administration. Suitable
carriers are
described in the most recent edition of Remington's Pharmaceutical Sciences, a
standard
reference text in the field, which is incorporated herein by reference.
Preferred examples of
such carriers or diluents include, but are not limited to, water, saline,
finger's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous
vehicles
such as fixed oils may also be used. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active compound, use
thereof in the
compositions is contemplated. Supplementary active compounds can also be
incorporated
into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with
its intended route of administration. Examples of routes of administration
include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation), transdermal
(i.e., topical), transmucosal, and rectal administration. Solutions or
suspensions used for
parenteral, intradennal, or subcutaneous application can include the following
components:
a sterile diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents; antibacterial agents
such as benzyl
alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate;
chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such
as acetates,
citrates or phosphates, and agents for the adjustment of tonicity such as
sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampoules, disposable
syringes or
multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration,
suitable carriers include physiological saline, bacteriostatic water,
Cremophor EL7M (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable
under the conditions of manufacture and storage and must be preserved against
the
contaminating action of microorganisms such as bacteria and fungi. The Garner
can be a
solvent or dispersion medium containing, for example, water, ethanol, polyol
(for example,
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glycerol, propylene glycol, and liquid polyethylene 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 the use of surfactants. Prevention of the action of
microorganisms can be
achieved by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases,
it will be
preferable to include isotonic agents, for example, sugars, polyalcohols such
as manitol,
sorbitol, sodium chloride in the composition. Prolonged absorption of the
injectable
compositions can be brought about by including in the composition an agent
which delays
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound
(e.g., a NOVX protein or anti-NOVX antibody) in the required amount in an
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
1 S compound into a sterile vehicle that contains a basic dispersion medium
and the required
other ingredients from those enumerated above. In the case of sterile powders
for the
preparation of sterile injectable solutions, methods of preparation are vacuum
drying and
freeze-drying that yields a powder of the active ingredient plus any
additional desired
ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible Garner. They
can
be enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral
therapeutic administration, the active compound can be incorporated with
excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also
be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid Garner is
applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible
binding agents, and/or adjuvant materials 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 colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring
agent such as peppermint, methyl salicylate, or orange flavoring.
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For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant,
e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barner to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, 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 against rapid elimination from the body, such as a
controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation
of such formulations will be apparent to those skilled in the art. The
materials can also be
obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (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 for the
subject to be
treated; each unit containing a predetermined quantity of active compound
calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical
Garner. 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.
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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 (see, e.g., U.S. Patent No.
5,328,470) or by
stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci.
ZISA 91:
S 3054-3057). The pharmaceutical preparation of the gene therapy vector can
include the
gene therapy vector in an acceptable diluent, 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 intact from recombinant cells, e.g.,
retroviral vectors, the
pharmaceutical preparation can include one or more cells that produce the gene
delivery
system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy
applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic
lesion in a
NOVX gene, and to modulate NOVX activity, as described further, below. In
addition, the
NOVX proteins can be used to screen drugs or compounds that modulate the NOVX
protein activity or expression as well as to treat disorders characterized by
insufficient or
excessive production of NOVX protein or production of NOVX protein forms that
have
decreased or aberrant activity compared to NOVX wild-type protein (e.g.;
diabetes
(regulates insulin release); obesity (binds and transport lipids); metabolic
disturbances
associated with obesity, the metabolic syndrome X as well as anorexia and
wasting
disorders associated with chronic diseases and various cancers, and infectious
disease(possesses anti-microbial activity) and the various dyslipidemias. In
addition, the
anti-NOVX antibodies of the invention can be used to detect and isolate NOVX
proteins
and modulate NOVX activity. In yet a further aspect, the invention can be used
in methods
to influence appetite, absorption of nutrients and the disposition of
metabolic substrates in
both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening
assays
described herein and uses thereof for treatments as described, supra.
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Screening Assays
The invention provides a method (also referred to herein as a "screening
assay") for
identifying modulators, i. e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or
have a
stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX
protein
activity. The invention also includes compounds identified in the screening
assays
described herein.
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
NOVX protein or polypeptide or biologically-active portion thereof. The test
compounds
of the invention can be obtained using any of the numerous approaches in
combinatorial
library methods known in the art, including: biological libraries; spatially
addressable
parallel solid phase or solution phase libraries; synthetic library methods
requiring
deconvolution; the "one-bead one-compound" library method; and synthetic
library
methods using affinity chromatography selection. The biological library
approach is
limited to peptide libraries, while the other four approaches are applicable
to peptide,
non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam,
1997.
Anticancef-I~rugDesign 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about S kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules. Libraries of
chemical and/or
biological mixtures, such as fungal, bacterial, or algal extracts, are known
in the art and can
be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in
the
art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. ZLS.A. 90:
6909; Erb, et al.,
1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 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. Ajagew. Chem. Int. Ed. Engl. 33: 2061;
and Gallop, et
al., 1994. J. Med. Clzem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechniques 13: 412-42I), or on beads (Lam, 1991. Nature 354: 82-84), on
chips (Fodor,
1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409),
spores (Ladner,
1 Ol



CA 02488547 2004-12-02
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U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Scierace 249: 386-390; Devlin,
1990.
Scieface 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:
6378-6382;
Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a
membrane-bound form of NOVX 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 NOVX protein determined. The cell, for example, can of mammalian origin
or a yeast
cell. Determining the ability of the test compound to bind to the NOVX 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 NOVX protein or
biologically-active portion thereof can be determined by detecting the labeled
compound in
a complex. 'For example, test compounds can be labeled with lzsI, sSS, laC, or
3H, either
directly or indirectly, and the radioisotope detected by direct counting of
radioemission or
by scintillation counting. Alternatively, test compounds can be enzymatically-
labeled with,
for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic
label detected by determination of conversion of an appropriate substrate to
product. In
one embodiment, the assay comprises contacting a cell which expresses a
membrane-bound
form of NOVX protein, or a biologically-active portion thereof, on the cell
surface with a
known compound which binds NOVX 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 NOVX protein, wherein determining the ability of the test compound to
interact with
a NOVX protein comprises determining the ability of the test compound to
preferentially
bind to NOVX protein or a biologically-active portion thereof as compared to
the known
compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of NOVX protein, or a biologically-active
portion
thereof, on the cell surface with a test compound and determining the ability
of the test
compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX
protein or
biologically-active portion thereof. Determining the ability of the test
compound to
modulate the activity of NOVX or a biologically-active portion thereof can be
accomplished, for example, by determining the ability of the NOVX protein to
bind to or
interact with a NOVX target molecule. As used herein, a "target molecule" is a
molecule
with which a NOVX protein binds or interacts in nature, for example, a
molecule on the
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surface of a cell which expresses a NOVX interacting protein, a molecule on
the surface of
a second cell, a molecule in the extracellular milieu, a molecule associated
with the internal
surface of a cell membrane or a cytoplasmic molecule. A NOVX target molecule
can be a
non-NOVX molecule or a NOVX protein or polypeptide of the invention. In one
embodiment, a NOVX target molecule is a component of a signal transduction
pathway
that facilitates transduction of an extracellular signal (e.g. a signal
generated by binding of
a compound to a membrane-bound NOVX molecule) through the cell membrane and
into
the cell. The target, for example, can be a second intercellular protein that
has catalytic
activity or a protein that facilitates the association of downstream signaling
molecules with
NOVX.
Determining the ability of the NOVX protein to bind to or interact with a NOVX
target molecule can be accomplished by one of the methods described above for
determining direct binding. In one embodiment, determining the ability of the
NOVX
protein to bind to or interact with a NOVX target molecule can be accomplished
by
determining the activity of the target molecule. For example, the activity of
the target
molecule can be determined by detecting induction of a cellular second
messenger of the
target (i.e. intracellular Gaz+, diacylglycerol, IP3, etc.), detecting
catalytic/enzymatic
activity of the target an appropriate substrate, detecting the induction of a
reporter gene
(comprising a NOVX-responsive regulatory element operatively linked to a
nucleic acid
encoding a detectable marker, e.g., luciferase), or detecting a cellular
response, for
example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay
comprising
contacting a NOVX protein or biologically-active portion thereof with a test
compound and
determining the ability of the test compound to bind to the NOVX protein or
biologically-active portion thereof. Binding of the test compound to the NOVX
protein can
be determined either directly or indirectly as described above. In one such
embodiment,
the assay comprises contacting the NOVX protein or biologically-active portion
thereof
with a known compound which binds NOVX 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 NOVX protein, wherein determining the ability of the test
compound to
interact with a NOVX protein comprises determining the ability of the test
compound to
preferentially bind to NOVX or biologically-active portion thereof as compared
to the
known compound.
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In still another embodiment, an assay is a cell-free assay comprising
contacting
NOVX protein or biologically-active portion thereof with a test eompound and
determining
the ability of the test compound to modulate (e.g. stimulate or inhibit) the
activity of the
NOVX protein or biologically-active portion thereof. Determining the ability
of the test
compound to modulate the activity of NOVX can be accomplished, for example, by
determining the ability of the NOVX protein to bind to a NOVX target molecule
by one of
the methods described above for determining direct binding. In an alternative
embodiment,
determining the ability of the test compound to modulate the activity of NOVX
protein can
be accomplished by determining the ability of the NOVX protein further
modulate a
NOVX target molecule. For example, the catalyticlenzymatic activity of the
target
molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically active portion thereof with a known compound which
binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test
compound, and
determining the ability of the test compound to interact with a NOVX protein,
wherein
determining the ability of the test compound to interact with a NOVX protein
comprises
determining the ability of the NOVX protein to preferentially bind to or
modulate the
activity of a NOVX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble
form or
the membrane-bound form of NOVX protein. In the case of cell-free assays
comprising the
membrane-bound form of NOVX protein, it may be desirable to utilize a
solubilizing agent
such that the membrane-bound form of NOVX protein is maintained in solution.
Examples
of such solubilizing agents include non-ionic detergents such as n-
octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton°,X-100, Triton° X-114,
Thesit°,
Isotridecypoly(ethylene glycol ether)n, N-dodecyl--N,N-dimethyl-3-ammonio-1-
propane
sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or .
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it
may
be desirable to immobilize either NOVX protein or its target molecule to
facilitate
separation of complexed from uncomplexed forms of one or both of the proteins,
as well as
to accommodate automation of the assay. Binding of a test compound to NOVX
protein, or
interaction of NOVX protein with a target molecule in the presence and absence
of a
candidate compound, can be accomplished in any vessel suitable for containing
the
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reactants. Examples of such vessels include microtiter plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be provided
that adds a
domain that allows one or both of the proteins to be bound to a matrix. For
example,
GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione
derivatized
microtiter plates, that are then combined with the test compound or the test
compound and
either the non-adsorbed target protein or NOVX protein, and the mixture is
incubated under
conditions conducive to complex formation (e.g., at physiological conditions
for salt and
pH). Following incubation, the beads or microtiter plate wells are washed to
remove any
unbound components, the matrix immobilized in the case of beads, complex
determined
either directly or indirectly, for example, as described, supra.
Alternatively, the complexes
can be dissociated from the matrix, and the level of NOVX protein binding or
activity
determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
1 S screening assays of the invention. For example, either the NOVX protein or
its target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g.,
biotinylation
kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated
96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX
protein or
target molecules, but which do not interfere with binding of the NOVX protein
to its target
molecule, can be derivatized to the wells of the plate, and unbound target or
NOVX protein
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 NOVX protein
or target
molecule, as well as enzyme-linked assays that rely on detecting an enzymatic
activity
associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in
a
method wherein a cell is contacted with a candidate compound and the
expression of
NOVX mRNA or protein in the cell is determined. The level of expression of
NOVX
mRNA or protein in the presence of the candidate compound is compared to the
level of
expression of NOVX mRNA or protein in the absence of the candidate compound.
The
candidate compound can then be identified as a modulator of NOVX mRNA or
protein
expression based upon this comparison. For example, when expression of NOVX
mRNA
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or protein is greater (i.e., statistically significantly greater) in the
presence of the candidate
compound than in its absence, the candidate compound is identified as a
stimulator of
NOVX mRNA or protein expression. Alternatively, when expression of NOVX 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
NOVX mRNA
or protein expression. The level of NOVX mRNA or protein expression in the
cells can be
determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX 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, et al., 1993. Gell 72: 223-232; Madura, et al., 1993. J. Biol. Glaem.
268:
12046-12054; Bartel, et al., 1993. Bi~teelZniques 14: 920-924; Iwabuchi, et
al., 1993.
Oncogene 8: 1693-1696; and Brent WO 94110300), to identify other proteins that
bind to or
interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX
activity. Such NOVX-binding proteins are also involved in the propagation of
signals by
the NOVX proteins as, for example, upstream or downstream elements of the NOVX
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 NOVX
is fused to a
gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-4). In
the other construct, a DNA sequence, from a library of DNA sequences, that
encodes an
unidentified protein ("prey" or "sample") is fused to a gene that codes for
the activation
domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to
interact, in vivo, forming a NOVX-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) that is operably linked to a
transcriptional
regulatory site responsive to the transcription factor. Expression of the
reporter gene can
be detected and cell colonies containing the functional transcription factor
can be isolated
and used to obtain the cloned gene that encodes the protein which interacts
with NOVX.
The invention further pertains to novel agents identified by the
aforementioned
screening assays and uses thereof for treatments as described herein.
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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. By way of example, and not of limitation, these sequences can be
used to: (i)
map their respective genes on a chromosome; and, thus, locate gene regions
associated with
genetic disease; (ii) identify an individual from a minute biological sample
(tissue typing);
and (iii) aid in forensic identification of a biological sample. Some of these
applications
are described in the subsections, below.
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. This
process is
called chromosome mapping. Accordingly, portions or fragments of the NOVX
sequences
of SEQ >D N0:2n-I, wherein n is an integer between 1 and 606, or fragments or
derivatives
thereof, can be used to map the location of the NOVX genes, respectively, on a
chromosome. The mapping of the NOVX sequences to chromosomes is an important
first
step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the NOVX sequences. Computer analysis of
the
NOVX, 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 containing the human gene corresponding to the
NOVX
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 mouse cells cannot grow, because they lack a
particular enzyme,
but in which 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 lines
can be established. Each cell line 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. See, e.g.,
D'Eustachio,
et al., 193. Science 220: 919-924. Somatic cell hybrids containing only
fragments of
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human chromosomes can also be produced by using human chromosomes with
translocations and deletions.
PCR mapping 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 NOVX sequences to design
oligonucleotide
primers, sub-localization can be achieved with panels of fragments from
specific
chromosomes.
Fluorescence ira 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 like 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.
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 panels of reagents can be
used for
marking multiple sites 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 sequence 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 are found, e.g., in McI~usick, MENDELIAN INHERITANCE IN MAN, available on-
line
through Johns Hopkins 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 ofphysically 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 NOVX gene, can be determined. If
a
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mutation is observed in some or all of the affected individuals but not in any
unaffected
individuals, then the mutation is likely to be the causative agent of the
particular disease.
Comparison of affected and unaffected individuals generally involves first
looking for
structural alterations in the chromosomes, such as deletions or translocations
that are
visible from chromosome spreads or detectable using PCR based on that DNA
sequence.
Ultimately, complete sequencing of genes from several individuals can be
performed to
confirm the presence of a mutation and to distinguish mutations from
polymorphisms.
Tissue Typing
The NOVX sequences of the invention can also be used to identify individuals
from
minute biological samples. In this technique, an individual's genomic DNA is
digested
with one or more restriction enzymes, and probed on a Southern blot to yield
unique bands
for identification. The sequences of the invention are useful as additional
DNA markers for
RFLP ("restriction fragment length polymorphisms," described in U.S. Patent
No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an
alternative
technique that determines the actual base-by-base DNA sequence of selected
portions of an
individual's genome. Thus, the NOVX sequences described herein can be used to
prepare
two PCR primers from the 5'- and 3'-termini 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 invention
can be used
to obtain such identification sequences from individuals and from tissue. The
NOVX
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. Much of the
allelic
variation is due to single nucleotide polymorphisms (SNPs), which include
restriction
fragment length polymorphisms (RFLPs).
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
can
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comfortably provide positive individual identification with a panel of perhaps
10 to 1,000
primers that each yield a noncoding amplified sequence of 100 bases. If coding
sequences,
such as those of SEQ ID N0:2n-1, wherein n is an integer between 1 and 606,
are used, a
more appropriate number of primers for positive individual identification
would be
500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which
diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring clinical trials
are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically.
Accordingly, one aspect of the invention relates to diagnostic assays for
determining
NOVX protein and/or nucleic acid expression as well as NOVX activity, in the
context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby determine
whether an
individual is afflicted with a disease or disorder, or is at risk of
developing a disorder,
associated with aberrant NOVX expression or activity. The disorders include
metabolic
disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated
cachexia,
cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's
Disorder, immune
disorders, and hematopoietic disorders, and the various dyslipidemias,
metabolic
disturbances associated with obesity, the metabolic syndrome X and wasting
disorders
associated with chronic diseases and various cancers. The invention also
provides for
prognostic (or predictive) assays for determining whether an individual is at
risk of
developing a disorder associated with NOVX protein, nucleic acid expression or
activity.
For example, mutations in a NOVX gene can be assayed in a biological sample.
Such
assays can be used for prognostic or predictive purpose to thereby
prophylactically treat an
individual prior to the onset of a disorder characterized by or associated
with NOVX
protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein,
nucleic acid expression or activity in an individual to thereby select
appropriate therapeutic
or prophylactic agents for that individual (referred to herein as
"pharmacogenomics").
Pharmacogenomics allows for 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 determine the ability of the individual
to respond to
a particular agent.)
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Yet another aspect of the invention pertains to monitoring the influence of
agents
(e.g., drugs, compounds) on the expression or activity of NOVX in clinical
trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of NOVX 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 NOVX
protein or
nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the
presence of NOVX is detected in the biological sample. An agent for detecting
NOVX
mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to
NOVX
mRNA or genomic DNA. The nucleic acid probe can be, for example,~a full-length
NOVX
nucleic acid, such as the nucleic acid of SEQ ID NO:2n-1, wherein n is an
integer between
1 and 606, or a portion thereof, such as an oligonucleotide of at least 15,
30, 50, 100, 250 or
500 nucleotides in length and sufficient to specifically hybridize under
stringent conditions
to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic
assays
of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
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
F(ab')a) can be used. The term "labeled", with regard to the probe or
antibody, is intended
to encompass direct labeling of the 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 NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as
well as
in vivo. For example, in vitro techniques for detection of NOVX mRNA include
Northern
hybridizations and in situ hybridizations. Ira vitr~ techniques for detection
of NOVX
protein include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence. In vitro techniques for
detection of
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NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques
for detection of NOVX protein include introducing into a subject a labeled
anti-NOVX
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.
In another embodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
capable of detecting NOVX protein, mRNA, or genomic DNA, such that the
presence of
NOVX protein, mRNA or genomic DNA is detected in the biological sample, and
comparing the presence of NOVX protein, mRNA or genomic DNA in the control
sample
with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a
biological sample. For example, the kit can comprise: a labeled compound or
agent
capable of detecting NOVX protein or mRNA in a biological sample; means for
determining the amount of NOVX in the sample; and means for comparing the
amount of
NOVX in the sample with a standard. The compound or agent can be packaged in a
suitable container. The kit can further comprise instructions for using the
kit to detect
NOVX protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant
NOVX 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 NOVX protein,
nucleic acid
expression or activity. Alternatively, the prognostic assays can be utilized
to identify a
subject having or at risk for developing a disease or disorder. Thus, the
invention provides
a method for identifying a disease or disorder associated with aberrant NOVX
expression
or activity in which a test sample is obtained from a subject and NOVX protein
or nucleic
acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX
protein or
nucleic acid is diagnostic for a subject having or at risk of developing a
disease or disorder
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associated with aberrant NOVX expression or activity. As used herein, a "test
sample"
refers to a biological sample obtained from a subject of interest. For
example, a test sample
can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether a subject can be administered an agent (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 NOVX expression or
activity. For
example, such methods can be used to determine whether a subject can be
effectively
treated with an agent for a disorder. Thus, the invention provides methods for
determining
whether a subject can be effectively treated with an agent for a disorder
associated with
aberrant NOVX expression or activity in which a test sample is obtained and
NOVX
protein or nucleic acid is detected (e.g., wherein the presence of NOVX
protein or nucleic
acid is diagnostic for a subject that can be administered the agent to treat a
disorder
associated with aberrant NOVX expression or activity).
1 S The methods of the invention can also be used to detect genetic lesions in
a NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a
disorder
characterized by aberrant cell proliferation and/or differentiation. In
various embodiments,
the methods include detecting, in a sample of cells from the subject, the
presence or
absence of a genetic lesion characterized by at least one of an alteration
affecting the
integrity of a gene encoding a NOVX-protein, or the misexpression of the NOVX
gene.
For example, such genetic lesions can be detected by ascertaining the
existence of at least
one of (i) a deletion of one or more nucleotides from a NOVX gene; (ii) an
addition of one
or more nucleotides to a NOVX gene; (iii) a substitution of one or more
nucleotides of a
NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration
in the '
level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification
of a
NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the
presence of
a non-wild-type splicing pattern of a messenger RNA transcript of a NOVX gene,
(viii) a
non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and
(x)
inappropriate post-translational modification of a NOVX protein. As described
herein,
there are a large number of assay techniques known in the art which can be
used for
detecting lesions in a NOVX gene. A preferred biological sample is a
peripheral blood
leukocyte sample isolated by conventional means from a subject. However, any
biological
sample containing nucleated cells may be used, including, for example, buccal
mucosal
cells.
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In certain embodiments, detection of the lesion involves the use of a
probe/primer in
a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
4,683,202),
such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain
reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et
al., 1994.
Proc. Natl. Aead. Sci. USA 91: 360-364), the latter of which can be
particularly useful for
detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl.
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 more primers that specifically
hybridize to a
NOVX gene under conditions such that hybridization and amplification of the
NOVX gene
(if present) occurs, and detecting the presence or absence of an amplification
product, or
detecting the size of the amplification product and 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
(see,
Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878),
transcriptional
amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTeclanology 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 are
present in very low
numbers.
In an alternative embodiment, mutations in a NOVX gene from a sample cell can
be
identified by alterations in restriction enzyme cleavage patterns. For
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.S. Patent No. 5,493,531) 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 NOVX can be identified by
hybridizing
a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays
containing
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hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al.,
1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example,
genetic
mutations in NOVX 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 changes between the sequences by making linear arrays of
sequential
overlapping probes. This step allows the identification of point mutations.
This is
followed by a second 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 NOVX gene and detect mutations by
comparing the
sequence of the sample NOVX with the corresponding wild-type (control)
sequence.
Examples of sequencing reactions include those based on techniques developed
by Maxim
and Gilbert, 1977. Proc. Natl. Acad. Sei. 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 (see, e.g.,
Naeve, et al.,
1995. Bioteclaraiques 19: 448), including sequencing by mass spectrometry
(see, e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechra~l. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNAIDNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In
general,
the art technique of "mismatch cleavage" starts by providing heteroduplexes of
formed by
hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded
duplexes are treated with an agent that cleaves single-stranded regions of the
duplex such
as which will exist due to basepair mismatches between the control and sample
strands.
For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with S1 nuclease to enzymatically digesting the mismatched regions. In
other
embodiments, either DNA/DNA or 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
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by size on denaturing polyacrylamide gels to determine the site of mutation.
See, e.g.,
Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al.,
1992. Methods
Enzymol. 217: 286-295. In an 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 NOVX cDNAs obtained from samples 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. See, e.g., Hsu, et al., 1994. Carcirrogeraesis
15: 1657-1662.
According to an exemplary embodiment, a probe based on a NOVX sequence, e.g.,
a
wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a
test
cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the
cleavage
products, if any, can be detected from electrophoresis protocols or the like.
See, e.g., U.S.
Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify mutations in NOVX genes. For example, single strand conformation
polymorphism (SSCP) may be used to detect differences in electrophoretic
mobility
between mutant and wild type nucleic acids. See, e.g., Orita, et al., 1989.
Proc. Natl. Acad.
Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992.
Genet. Araal.
Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX
nucleic acids will be denatured and allowed to renature. The secondary
structure of
single-stranded nucleic acids varies according to sequence, 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 probes. 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 one embodiment, the subject method
utilizes
heteroduplex analysis to separate double stranded heteroduplex molecules on
the basis of
changes in electrophoretic mobility. See, e.g., 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). See, e.g., Myers, et al., 1985. Nature
313: 495.
When 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
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high-melting GC-rich DNA by PCR. In a further embodiment, a temperature
gradient is
used in place of a denaturing gradient to identify differences in the mobility
of control and
sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chenz. 265:
12753.
Examples of other techniques for detecting point mutations include, but are
not
limited to, selective oligonucleotide hybridization, selective amplification,
or selective
primer extension. For example, oligonucleotide primers may be prepared in
which the
known mutation is placed centrally and then hybridized to target DNA under
conditions
that permit hybridization only if a perfect match is found. See, e.g., 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 that 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 molecule (so that amplification depends on differential hybridization;
see, e.g., Gibbs,
et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of
one primer
where, under appropriate conditions, mismatch can prevent, or reduce
polymerase
extension (see, e.g., 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. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is
anticipated that in
certain embodiments amplification may also be performed using Taq ligase for
amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In
such cases,
ligation will occur only if there is a perfect match at the 3'-terminus of the
5' sequence,
making it possible to detect the presence of a known mutation at a specific
site by looking
for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing
pre-packaged diagnostic kits comprising at least one probe nucleic acid or
antibody reagent
described herein, which may be conveniently used, e.g., in clinical settings
to diagnose
patients exhibiting symptoms or family history of a disease or illness
involving a NOVX
gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes,
in
which NOVX is expressed may be utilized in the prognostic assays described
herein.
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However, any biological sample containing nucleated cells may be used,
including, for
example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity
(e.g., NOVX gene expression), as identified by a screening assay described
herein can be
administered to individuals to treat (prophylactically or therapeutically)
disorders. The
disorders include but are not limited to, e.g., those diseases, disorders and
conditions listed
above, and more particularly include those diseases, disorders, or conditions
associated
with homologs of a NOVX protein, such as those summarized in Table A.
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 the 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 the
individual's
genotype. Such pharmacogenomics can further be used to determine appropriate
dosages
and therapeutic regimens. Accordingly, the activity of NOVX protein,
expression of
NOVX nucleic acid, or mutation content of NOVX 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., Eichelbaum, 1996. Clin. Exp. Pharmacol. Playsiol., 23: 983-985;
Linden 1997.
Clin. Claem., 43: 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 (altered drug action) or genetic conditions transmitted as single
factors altering
the way the body acts on drugs (altered drug metabolism). These
pharmacogenetic
conditions can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited
enzymopathy in which the main clinical complication is hemolysis after
ingestion of
oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of
fava beans.
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As an illustrative 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 pregnancy zone protein precursor 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 polymorphisms are expressed in two phenotypes in the
population, the
extensive metabolizes (EM) and poor metabolizes (PM). The prevalence of PM is
different
among different populations. For example, the gene coding for CYP2D6 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 CYP2C 19 quite
frequently experience exaggerated drug response and side effects when they
receive
standard doses. If a metabolite is the active therapeutic moiety, PM show no
therapeutic
response, as demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. At 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 activity of NOVX protein, expression of NOVX nucleic acid, or
mutation
content of NOVX genes in an individual can be determined to thereby select
appropriate
agents) for therapeutic or prophylactic treatment of the individual. In
addition,
phannacogenetic 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 NOVX modulator, such as a modulator identified
by one of
the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of NOVX (e.g., the ability to modulate aberrant cell proliferation
and/or
differentiation) can be applied not only in basic drug screening, but also in
clinical trials.
For example, the effectiveness of an agent determined by a screening assay as
described
herein to increase NOVX gene expression, protein levels, or upregulate NOVX
activity,
can be monitored in clinical trails of subjects exhibiting decreased NOVX gene
expression,
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protein levels, or downregulated NOVX activity. Alternatively, the
effectiveness of an
agent determined by a screening assay to decrease NOVX gene expression,
protein levels,
or downregulate NOVX activity, can be monitored in clinical trails of subjects
exhibiting
increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such
clinical trials, the expression or activity of NOVX and, preferably, other
genes that have
been implicated in, for example, a cellular proliferation or immune disorder
can be used as
a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule)
that modulates NOVX activity (e.g., identified in a screening assay as
described herein) can
be identified. Thus, to study the effect of agents on cellular proliferation
disorders, for
example, in a clinical trial, cells can be isolated and RNA prepared and
analyzed for the
levels of expression of NOVX 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, or alternatively by measuring the amount of
protein
produced, by one of the methods as described herein, or by measuring the
levels of activity
of NOVX or other genes. In this manner, the gene expression pattern can serve
as a
marker, indicative of the physiological response of the cells to the agent.
Accordingly, this
response state may be determined before, and at various points during,
treatment of the
individual with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness of treatment of a subject with an agent (e.g., an agonist,
antagonist, protein,
peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified by
the screening assays described herein) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of the agent;
(ii) detecting
the level of expression of a NOVX protein, mRNA, or genomic DNA 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 NOVX
protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the level of
expression or
activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration
sample
with the NOVX protein, mRNA, 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 NOVX to higher levels than detected, i.e., to increase the
effectiveness of the
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agent. Alternatively, decreased administration of the agent may be desirable
to decrease
expression or activity of NOVX to lower levels than detected, i. e., to
decrease the
effectiveness of the agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of
treating a
subject at risk of (or susceptible to) a disorder or having a disorder
associated with aberrant
NOVX expression or activity. The disorders include but are not limited to,
e.g., those
diseases, disorders and conditions listed above, and more particularly include
those
diseases, disorders, or conditions associated with homologs of a NOVX protein,
such as
those summarized in Table A.
These methods of treatment will be discussed more fully, below.
Diseases and Disorders
Diseases and disorders that are characterized by increased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics
that antagonize
activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that
may be utilized include, but are not limited to: (i) an aforementioned
peptide, or analogs,
derivatives, fragments or homologs thereof; (ii) antibodies to an
aforementioned peptide;
(iii) nucleic acids encoding an aforementioned peptide; (iv) administration of
antisense
nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a
heterologous insertion
within the coding sequences of coding sequences to an aforementioned peptide)
that are
utilized to "knockout" endogenous function of an aforementioned peptide by
homologous
recombination (see, e.g., Capecchi, 1989. Sciefice 244: 1288-1292); or (v)
modulators ( i.e.,
inhibitors, agonists and antagonists, including additional peptide mimetic of
the invention
or antibodies specific to a peptide of the invention) that alter the
interaction between an
aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that
upregulate
activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that
may be utilized include, but are not limited to, an aforementioned peptide, or
analogs,
derivatives, fragments or homologs thereof; or an agonist that increases
bioavailability.
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Increased or decreased levels can be readily detected by quantifying peptide
and/or
RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and
assaying it in vitro
for RNA or peptide levels, structure and/or activity of the expressed peptides
(or mRNAs
of an aforementioned peptide). Methods that are well-known within the art
include, but are
not limited to, immunoassays (e.g., by Western blot analysis,
immunoprecipitation
followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,
immunocytochemistry, etc.) and/or hybridization assays to detect expression of
mRNAs
(e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a
disease
or condition associated with an aberrant NOVX expression or activity, by
administering to
the subject an agent that modulates NOVX expression or at least one NOVX
activity.
Subjects at risk for a disease that is caused or contributed to by aberrant
NOVX expression
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 characteristic of the NOVX aberrancy,
such that a
disease or disorder is prevented or, alternatively, delayed in its
progression. Depending
upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX
antagonist
agent can be used for treating the subject. The appropriate agent can be
determined based
on screening assays described herein. The prophylactic methods of the
invention are
further discussed in the following subsections.
Therapeutic Methods
Another aspect of the invention pertains to methods of modulating NOVX
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
NOVX protein activity associated with the cell. An agent that modulates NOVX
protein
activity can be an agent as described herein, such as a nucleic acid or a
protein, a
naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX
peptidomimetic, or other small molecule. In one embodiment, the agent
stimulates one or
more NOVX protein activity. Examples of such stimulatory agents include active
NOVX
protein and a nucleic acid molecule encoding NOVX that has been introduced
into the cell.
In another embodiment, the agent inhibits one or more NOVX protein activity.
Examples
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of such inhibitory agents include antisense NOVX nucleic acid molecules and
anti-NOVX
antibodies. These modulatory methods can be performed ira vitro (e.g., by
culturing the cell
with the agent) or, alternatively, in vivo (e.g., by administering the agent
to a subject). As
such, the invention provides methods of treating an individual afflicted with
a disease or
disorder characterized by aberrant expression or activity of a NOVX protein or
nucleic acid
molecule. In one embodiment, the method involves administering an agent (e.g.,
an agent
identified by a screening assay described herein), or combination of agents
that modulates
(e.g., up-regulates or down-regulates) NOVX expression or activity. In another
embodiment, the method involves administering a NOVX protein or nucleic acid
molecule
as therapy to compensate for reduced or aberrant NOVX expression or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is
abnormally downregulated andlor in which increased NOVX activity is likely to
have a
beneficial effect. One example of such a situation is where a subject has a
disorder
characterized by aberrant cell proliferation and/or differentiation (e.g.,
cancer or immune
associated disorders). Another example of such a situation is where the
subject has a
gestational disease (e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays
are
performed to determine the effect of a specific Therapeutic and whether its
administration
is indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with
representative cells of the types) involved in the patient's disorder, to
determine if a given
Therapeutic exerts the desired effect upon the cell type(s). Compounds for use
in therapy
rnay be tested in suitable animal model systems including, but not limited to
rats, mice,
chicken, cows, monkeys, rabbits, and the like, prior to testing in human
subjects. Similarly,
for in vivo testing, any of the animal model system known in the art may be
used prior to
administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
The NOVX nucleic acids and proteins of the invention are useful in potential
prophylactic and therapeutic applications implicated in a variety of
disorders. The
disorders include but are not limited to, e.g., those diseases, disorders and
conditions listed
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above, and more particularly include those diseases, disorders, or conditions
associated
with homologs of a NOVX protein, such as those summarized in Table A.
As an example, a cDNA encoding the NOVX protein of the invention may be
useful in gene therapy, and the protein may be useful when administered to a
subject in
S need thereof. By way of non-limiting example, the compositions of the
invention will have
efficacy for treatment of patients suffering from diseases, disorders,
conditions and the like,
including but not limited to those listed herein.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of
the invention, or fragments thereof, may also be useful in diagnostic
applications, wherein
the presence or amount of the nucleic acid or the protein are to be assessed.
A further use
could be as an anti-bacterial molecule (i. e., some peptides have been found
to possess
anti-bacterial properties). These materials are further useful in the
generation of antibodies,
which immunospecifically-bind to the novel substances of the invention for use
in
therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do
not
limit the scope of the invention described in the claims.
FXAMPT.F~
Example A: Polynucleotide and Polypeptide Sequences, and Homology Data
Example 1.
The NOV 1 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table lA.
Table lA. NOVl Sequence Analysis I
NOVla, CG191083-O1 _SEQ_m NO: 1 4494 by __ ___
DNA Sequence_ ORF S_ta_rt: ATG at~59~ O12F Stop: TA_A at 1_394_
CGAGAGCGGGAGTGAGCGAGCGAGCGAGTTGCCGAGCGCGCCCCGTCCCTCGCGCGCGATGCTCCCC
CCTGAGCTTGCGGCTGGCGCTGGCGCGGAGCGGCGCAGAGCGCGGTCC
CCAGCATCAGCCCCCCGAGGGGACCTGATGTTCCTGCTGGACAGCTCAGCCAGCGTCTCTCACTACG
ATGCCAAGGAACAGCTGTTTGCTGAAGCATCAGGTGCCCGGCCAGGGGTGCCCAAAGTGCTG
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TTGTCCAAGAGCTGAGGGGC
TTCTCGACGCGATGCGGCCGCAGCAGCTCCATGCCACGGAGATCACGTCCAGCGGCTTCCGCCT
TCCTGCG
TCGTCATCTCCCACGCC
G
CAGTTCGGGCCGCTGCGGGGCGGGGAGGCGCAGCGGGTGGAGGTGCCCGCGGGCCGCAACTGCACCA
C
CCGGCCTTTCCCCACGCGGACTCCGCGCGACCCCGGCCCTCTCCCTGCGGCCGCAGGGCTTCCCCGC
C
CCCAGAGCCGGGCGTCGTGTGGGTCCGTGGGTGATAATTGAGAGCGTCAGACCCAGGACTGTTCAGG
CACGTGTGAAGACCGGGCCCCAAGTGGCAAGGGCTGGCCTGGGGCGG
AGGAAGCGGAAGGGGAATCGCGGGAAGCTGGCCCAGGTCAGGTC
C
TGCCCAGGACACCAGCTGG
CCGCCCACCGCCACTATCAGGCCCCGGGACCGCACTGACAGGAAACCTTCCGTCGTGA
TCTTCT
ACTCACTGCCCTGGGGTCCGTGGGCAAGT
TGC
TTCAGTAAATCAGATGTACACCGAGCCT
TGCTTTACTCCTCCACAAACAGCAGCCGCCGACAGGACAGCTTGTGAACGTCT
G
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CAGGGCCACGCGGGCTCCCTCTGCTGGCAAAGCCACACCCTGCACCGCATGGGGT
C_
CTCCCTCAGGTCTGGTGGGCATTGTGCCAGGGGCCACCTTCGGGACAGCTGGCCTTGGCACCGTCTC
C_
CAAGAACCCAGCTCTGTGCATCCCGTGGCCCCACCAGAAACCCAAGGCCACACCGCCCACCAGCTGG
TGGAGGGCCGGCTGGGGCCAGGCTCACCACGCAGAGTTTGCCCTCCGGTGTG
GCTGGACTTTGGGTTTCTCCTGAGCTCCCCTTAGCCCCCAAGTCAGGCCACGGAGGCCTGAGTAGGA
C_
CCATGGGCTGTGTGTGTCACTGCAGGTGGCGTGCTCACAACTGCACTGCTGGTCGGCAGGTGGCCAG
GTTGCAGGCCCGGGCCTCGGGCCCCACCTCCTCTGGAGGGGTAGGATCTCCTTCGTGCAGCCCCTGC
C_
CCCTACCCTGTTCTCCGGAGTGTTGGCAGATCTGAGCCCACGGTCACGTGAGAGGAGATGCCTCTTG
C
T_
GGTGGCCTCAGTGGCTTCTGTGGACGACTCAGACTCACATGAGAAGCTGGGAGGAGCTCCAGCTCTG
_C
AATCCCGAGAGGGCAGAGCGGGGGCCCTGGCCACAACCCTGCTCCCCACATACCCGTCTGGCAGAGG
_C
TCTGCCTCCCCTCTGCCCATCTCTGCACACCAAGGCTGAGCACAGCACAAGGCCTCACGGAGAGGGT
G
G
TCTGCACCAAGTCTCAGAACAGCTCTG
AATCCTGGCCCCGACTCAGTCCACCCAGGGTGCAGTGCAGAGGCTGATACCCGCCAGGACTTCCTTG
_C
CAAGGGCCCGGCCACGTCACAAGGCCACTGCACGCTTTTCGACATGCACCTGGAAATCGGGAAGGGC
C
T_
CCCACACCTGCGCCAGTTTCCACCCTCTCTGTGAGCAGGGCTGCGGTCACCTCCCACATCTGAAGAG
CTGGACAGCCCATTGCTGGCCACTGGACGGAGAGGCAGAGGGGGCTGAAATTCGGGCCCATGCCTCT
TGAGCGATGACGGAGCAACAGCTCTCCAGCACGTGAAGCTCTCCAGACAGCTGTTCGTGAGAAGCCA
TTTCTGGGGAGTGGGGTGTCCAAGCGTGGGCCACGCTGCTG
AATAAACCAAGTCAATTTTCTATA
a, CG191083-O1 ~SEQ m NO: 2 445 as MW at 46803.S1~D
iSequence
PPASAPRGDLMFLLDSSASVSHYEFSRVREFVGQLVAPLPLG
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VHVGSRPYTEFPFGQHSSGEAAQDAVRASAQRMGDTHTGLALVYAKEQLFAEASGARPGV
VWVTDGGSSDPVGPPMQELI~DLGVTVFIVSTGRGNFhELSAAASAPAEKHLHFVDVDDLHIIVQ
ILDAMRPQQLHATEITSSGFRLAWPPLLTADSGYYVLELVPSAQPGAARRQQLPGNATDWIWA
VPESNVRLLRPQILRVRTRPGEAGPGASGPESGAGPAPTQLAALPAPEEAGPERIV
II
LRVSWAPALGSAAALGYHVQFGPLRGGEAQRVEVPAGRNCTTLQGLAPGTAYLV'I'VTAAF
SGRESALSAKACTPDGPRPRPRPVPRAPTPGTASREP
Further analysis of the NOV 1 a protein yielded the following properties shown
in Table 1B.
Table 1B. Protein Sequence Properties NOVla
SignalP analysis: ~ Cleavage site between residues 19 and 20
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 0; pos.chg 0; neg.chg 0
H-region: length 13; peak value 8.36
PSG score: 3.96
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 2.40
possible cleavage site: between 18 and 19
» > Seems to have a cleavable signal peptide (1 to 18)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 19
Tentative number of TMS(s) for the threshold 0.5: 0
number of TMS(s) .. fixed
PERIPHERAL Likelihood = 2.17 (at 60)
ALOM score: 2.17 (number of TMSs: 0)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 9
Charge difference: 1.0 C( 2.0) - N( 1.0)
C > N: C-terminal side will be inside
» >Caution: Inconsistent mtop result with signal peptide
MITDISC: discrimination of mitochondrial targeting seq
R content: 2 Hyd Moment(75): 2.90
Hyd Moment(95): 1.31 G content: 2
D/E content: 1 S/T content: 3
Score: -4.22
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at 29 ARS~GA
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
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content of basic residues: 9.~%
NLS Score: -0.47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 7l PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 76.7
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
33.3 %: extracellular, including cell wall
33.3 %: mitochondrial
22.2 %: vacuolar
11.1 %: endoplasmic reticulum
» prediction for CG191083-01 is exc (k=9)
A search of the NOV Z a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 1 C.
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Table 1C. Geneseq Results
for NOVla


NOVl Identities/
a


Geneseq Protein/Organism/Length Residues!SimilaritiesExpect
for


Identifier[Patent #, Date] Match the Matched Value


ResiduesRegion


ABP69674 Human polypeptide SEQ 1..445 445/445 (100%)0.0
ID NO


1721 - Homo Sapiens, 445 I..445 445/445 (100%)
aa.


[W0200270539-A2, 12-SEP-2002]


AAE32502 Human Willebrand Factor 1..445 418/445 (93%)0.0
A


domain related-protein 1..418 418/445 (93%)
(WARP) -


Homo Sapiens, 418 aa.


[W0200288184-Al, 07-NOV-


2002]
i


AAE32501 Mouse Willebrand Factor 1..445 324/445 (72%)0.0
A


domain related-protein 1..415 356/445 (79%)
(WARP) -


Mus sp, 415 aa. [W0200288184-


A1, 07-NOV-2002]


AAW86326 Kidney injury associated 1..445 325/445 (73%)e-179
molecule


HW059 protein - Rattus 7..421 356/445 (79%)
sp, 421 aa.


[WO9853071-Al, 26-NOV-1998]


~""
AAB42581 ~ Human ORFX ORF2345 3..296 274/294 (93%)e-150


polypeptide sequence SEQ 10..299 274/294 (93%)
ID


N0:4690 - Homo Sapiens,
299 aa.


[WO200058473-A2, 05-OCT-


2000]


In a BLAST
search
of public
sequence
databases,
the NOV
1 a protein
was found
to have


homology
to the
proteins
shown
in the
BLASTP
data
in Table
1D.


Table 1D. Public BLASTP
Results for NOVla


Protein NOVla Identities/


AccessionProtein/Organism/Length Residues/SimilaritiesExpect
for


Number Match the Matched Value


ResiduesPortion


Q8R2Z5 Hypothetical protein (VON1..445 325/445 (73%)e-180


WILLEBRAND factor A-related1..415 356/445 (79%)


protein homology - Mus
musculus


(Mouse), 415 aa.


Q8COQ7 VON WILLEBRAND factor 1..445 324/445 (72%)e-180
A-


related protein homolog 1..415 355/445 (78%)
- Mus


musculus (Mouse), 415 ~
aa.


Q923K3 Von Willebrand factor 1..445 324/445 (72%)e-180
A-related


protein - Mus musculus 1..415 356/445 (79%)
(Mouse),


415 aa.


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CAC39705 Sequence 47 from Patent 1..257 253/257 (98%)e-142


EP1067182 - Homo sapiens 1..257 254/257 (98%)


(Human), 285 aa.


Q9H6J5 Hypothetical protein FLJ22215 233/233 (100%)e-134
- 213..445


Homo sapiens (Human), 233 aa. 233/233 (100%)
1..233


PFam analysis
predicts
that
the NOV
1 a protein
contains
the domains
shown
in the
Table


1 E.


Table lE. Domain Analysis of
NOVla


Identities/


Pfam Domain NOVla Match Region SimilaritiesExpect Value


for the Matched Region


vwa 34..205 68/194 (35%) 1.5e-40


132/194 (68%)


fn3 212..287 22/85 (26%) 0.18


52/85 (61 %)


fn3 332..413 30/85 (35%) 1.4e-11


53/85 (62%)



Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 2A.
Table 2A. NOV2 Sequence Analysis
NOV2a, ~CG191745-O1 SEQ )D N0. 3 3462 by
q ,.~~...Start. ATG at 2~..~_
~,.~............_...,...................................
DNA Se uence ; v ~ ~ ~ ~ ~ ~ ~ ~ O~ Stop: TGA at 3443
TATTTTCCGGTCCTTCAAG
TGCAGTGCAGCAGTTTATAAATCCAAAAGGCAACTGGTTACTGGTTGGTTCAC
ATCCACTGCC
TGTGAAAAACTAAATTTGCAAACTTCAACAAGCATTCCAAATGTTACTGAGATGAAAACCAACAT
C
TCAGTATTACACAACGGGTGTGTGTTCTGACATCAGTCCTGATTTTCAGCTC
CAGCCAGCTTCTCACCTGCAACTCAGCCCTGCCCTTCCCTCATAGATGTTGTGGTTGTGTGTGATGA
TA
TAATCCAAGAGTTGTGTTTAACTTG
C
TATGCTTATTCAGCAGCTTCTGGTGGGCGAC
TGGTTCAATGTTGAAAGCT
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TTTAATAAAAGAAATAAAAGCAATCGCTAGTATTCCAACAGAAAGAT
ACTGTTCAAGGAGGAGACAACTTTCAGATGGAAATGTCACAAGTGGGATTCAGTGC
TATTCTGATGCTGGGTGCAGTGGGAGCTTTTGGCTGGAGTGGGACCA
TCTCATGGCCATTTGATCTTTCCTAAACAAGCCTTTGACCAAATTCTGCAGGAC
TTATACCGGCCAGATAGTGCTATATAGTGTGAATGAGAATGGCAATA
ACATGAGTGACCTAAA
TCAAAGAGGGCATTTTGGGTCAGCACCAATTTCTTG
TTTGGTTCAGCAATTGCAGCTCTTTCAGACATCAACATG
TTCTGGAGCTGTATACATTTA
TGGTCATCAGGGCACTATCCGCACAAAGTATTCCCAGAAAATCTTGGGATCCGATGGAGCCTTTA
G
TGGCTATGGAGATTTAAATGGGGATTCCATCACC
G
GAAGCTTCATTCACACCAGAAAAAATCACTTTGGTCAACAAGAATGCTCAGATAATTCTCAAACTCT
G
CTTCAGTGCAAAGTTCAGACCTACTAAGCAAAACAATCAAGTGGCCATTGTATATAACATCACACTT
G
ATGCAGATGGATTTTCATCCAGAGTAACCTCCAGGGGGTTATTTAAAGAAAACAATGAAAGGTGCCT
G
CAGAAGAATATGGTAGTAAATCAAGCACAGAGTTGCCCCGAGCACATCATTTATATACAGGAGCCCT
TTCCTTTCCACAAAGACTGTGGTGAGGACGG
TTTCTGATCTAGTCCTAGATGTCCGACAAATACCAGCTGCTCAAGAACAACCCTTTATTG
TAAAAGGGAAAGTGCATACAAC
TGTAGGCTACCCTGCTTTAA
TTAACTTTGACTTCAATCTTCAAAACCTTCAGAATCAGGCG
TAATTTGGTCAACCTCAA
TGATGCTGAAATTCACTTAACAAAGGTAACAACAGGAAGTGTTCCAGTAAGCA
TCCACATCCCTCAGTATACCAAAGAAAAGAACCCACTGATGTACCTAACTGGG
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TAGGACAAAC
TCTTCTTCTGTATCTTTCAAAAGTGAAAATTTCAGGCACACCAAAGAATTGAACTGCAGAACTGCTT
TGTTACCTGCTGGTTGAAAGACGTTCACATGAAAGGAGAATACTTTGTTAATGTGACT
TGATAA
CGAAGTACCAACAGGAGTTATAATAGGAAGTATAATTGCTGGAATCCTT
TTTTATGGAAGCTCGGCTTCTTCAAAAGAAAATATGAAAAGATGAC
TTGATGAGACCACAGAGCTCAGTAGCTGAACCAGCAGACCTACCTG
2a, Cq 191745-O1 ~SEQ >D NO: 4 l 147 as -~MW at 125495.9kD
in Se uence _ _ ___ _ __
l2TGAAPLPLLLVLALSQGILNCCLAYNVGLPEAKIFSGPSSEQFGYAVQQFINPKGNWLLVGS
TATCEKLNLQTSTSIPNVTEMKTNMSLGLILTRNMGTGGFLTCGPLW
ISPDFQLSASFSPATQPCPSLIDVVWCDESNSIYPWDAVKNFLEKFVQGLD
IVATSQTSQYGGDLTNTFGAIQYARKYAYSAASGGR
SATKVMVWTDGESHDGSMLKAVIDQCNHDNILRFGIAVLGYLNRNALDTKNLIKEIKAIASIPTER
FFNVSDEAALLEKAGTLGEQIFSIEGTVQGGDNFQMEMSQVGFSADYSSQNDILMLGAVGAFGWSGT
FPKQAFDQILQDRNHSSYLGYSVAAISTGESTHFVAGAPRANYTGQIVLYSVNENGN
QAHRGDQIGSYFGSVLCSVDVDKDTITDVLLVGAPMYMSDLKKEEGRVYLFTIKEGILGQHQFL
IENTRFGSAIAALSDINMDGFNDVIVGSPLENQNSGAVYIYNGHQGTTRTKYSQKILGSDGAF
SHLQYFGRSLDGYGDLNGDSITDVSIGAFGQVVQLWSQSIADVAIEASFTPEKITLVNKNAQIILKL
C
FSAKFRPTKQNNQVAIVYNITLDADGFSSRVTSRGLFKENNERCLQKNMVVNQAQSCPEHIIYIQEP
S
DVVNSLDLRVDISLENPGTSPALEAYSETAKVFSIPFHKDCGEDGLCISDLVLDVRQIPAAQEQPFI
SAYNTGIVVDFSENLFFASFSLPVDGTEVTCQVAASQKSVACDVGYPAL
LSFQALSESQEENKADNLVNLKIPLLYDAEIHLTKVTTGSVPVS
ATVIIHIPQYTKEKNPLMYLTGVQTDKAGDISCNADINPLKIGQTSSSVSFKSENFRHTKELNCRTA
S
CSNVTCWLKDVHMKGEYFVNVTTRIWNGTFASSTFQTVQLTAAAEINTYNPEIYVIEDNTVTIPLMI
IGSIIAGILLLLALVAILWKLGFFKRKYEKMTKNPDEIDETTELSS
Further analysis of the NOV2a protein yielded the following properties shown
in Table 2B.
Table 2B. Protein Sequence Properties NOV2a
SignalP analysis: Cleavage site between residues 30 and 31
PSORT II analysis:
PSG: a new signal peptide prediction method
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N-region: length 5; pos.chg 1; neg.chg 1
H-region: length 30; peak value 9.82
PSG score: 5.42
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): 1.12
possible cleavage site: between 22 and 23
» > Seems to have a cleavable signal peptide (1 to 22)
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 23
Tentative number of TMS(s) for the threshold 0.5: 2
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood =-13.27 Transmembrane 1100 -1116'
PERIPHERAL Likelihood = 0.95 (at 943)
ALOM score: -13.27 (number of TMSs: l)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 11
Charge difference: -2.0 C(-1.0) - N( 1.0)
N >= C: N-terminal side will be inside
» > membrane topology: type 1a (cyt0plasmic tail 1117 to 1147)
MITDISC: discrimination of mitochondrial targeting seq
R content: 1 Hyd Moment(75): 10.10
Hyd Moment(95): 5.95 G content: 4
D/E content: 2 S/T content: 2
Score: -6.90
Gavel: prediction of cleavage sites for mitochondrial preseq
R-2 motif at l5 ERT~GA
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 7.7~
NLS Score: -0.47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: GPER
;none
'SKL: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
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Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tai1:1128
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 7l PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: cytoplasmic
Reliability: 89
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
55.6 %: endoplasmic reticulum
22.2 %: Golgi
11.1 %: plasma membrane
11.1 %: extracellular, including cell wall
» prediction for CG191745-01 is end (k=9)
A search of the NOV2a protein against the Geneseq database, a proprietary
database that
contains sequences published in patents and patent publication, yielded
several homologous
proteins shown in Table 2C.
",~""",-~u"~y~~~,WU"~~ Table 2C. Geneseq Results for NOV2a
W.~"-..."W.,.~.,.W""~..~.....,~~,;.~,..~..~..... ~_.~.,.".."..~.,~. .~."
NOV2a Y~ Identities/
Geneseq Protein/Organism/Length Residues/ Similarities for Expect
Identifier [Patent #, Date) Match the Matched Value
Residues Region
AAG79775 ~ Alpha2 integrin -Homo sapiens, l..l 147 1146/1181 (97%) 0.0
1181 aa. [W02002101070-A2, 19- ~ 1..1181 I 147/1181 (97%)
DEC-2002]
ABU03548 Angiogenesis-associated human 1..1147 1146/1181 (97%) 0.0
1..1181 1147/1181 (97%)
134



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sapiens, 1181 aa. [W0200279492-~


A2, 10-OCT-2002]


ABU03617 Human expressed protein l..l 1146/1181 0.0
tag 147 (97%)


(EPT) #283 - Homo Sapiens,1..1181 1147/1181
1181 (97%)


aa. [W0200278524-A2,
10-OCT-


2002]


ABU03616 Human expressed protein 1..1147 1146/1181 0
. tag (97%) 0


(EPT) #282 - Homo Sapiens,1..1181 1147/1181 .
1181 (97%)


aa. [W0200278524-A2,
10-OCT-


2002]


ABU03614 Human expressed protein 1..1147 1146/1181 0.0
tag (97%)


(EPT) #280 - Homo Sapiens,l..l 1147/1181
1181 181 (97%)


aa. [W0200278524-A2,
IO-OCT-


2002]


In a BLAST
search
of public
sequence
databases,
the NOV2a
protein
was found
to have


homology ata in
to the Table
proteins 2D.
shown
in the
BLASTP
d


Table
2D. Public
BLASTP
Results
for NOV2a


Protein NOV2a Identities/
:


AccessionProtein/Organism/Length Residues/Similarities
Expect
for


Number Match the Matched Value


ResiduesPortion
'


P17301 Integrin 1..1147 1146/1u181 0.0
alpha-2 a (97%)
precursor
(Platelet


membrane l..l 1147/1181
glycoprotein 181 (97%)
Ia)
(GPIa)


(Collagen
receptor)
(VLA-2
alpha


chain)
(CD49b)
-
Homo
sapiens


(Human),
1181
aa.


P53710 Integrin 12..1147986/1170 (84%)0.0
alpha-2 '
precursor
(Platelet
~


membrane 1..1170 1069/1170
glycoprotein (91%)
Ia)
(GPIa)
'


(Collagen
receptor)
(VLA-2
alpha


chain)
(CD49b)
-
Bos
taurus


(Bovine),
1170
as
(fragment).


Q62469 Integrin l..l 945/1181 (80%)0.0
alpha-2 147
precursor
(Platelet


membrane 1..1178 1040/1181
glycoprotein (88%)
Ia)
(GPIa)


(Collagen
receptor)
(VLA-2
alpha


chain)
(CD49b)
-
Mus
musculus


(Mouse),
1178
aa.


.~...W....~..,.~...
PI8614 Integrin 1..1131 471/1202 (39%)0.0
alpha-I
precursor
(Laminin
~


and 1..1176 678/1202 (56%)
collagen
receptor)
(VLA-1)


(CD49a)
-
Rattus
norvegicus
(Rat),


1180
aa.


042094 ALPHA1 29..113145611179 (38%)0
integrin 0
-
Gallus
gallus


(Chicken), 17..1167671/1179 (56%).
1171 '
aa.


135



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PFam analysis
predicts
that the
NOV2a protein
contains
the domains
shown in
the Table


2E.
~... .......
.


~ Table 2E. .... . ..................
Domain Analysis . .
of NOV2a.......
_


Identities/


Pfam DomainNO V2a Match Similarities Expect Value
Region


for the Matched
Region


vwa 174..361 64/203 (32%) l.le-47


146/203 (72%)


FG-GAP 491..554 18/64 (28%) 0.00012


52/64 (81 %)


FG-GAP 555..602 14/52 (27%) 0.00062


37/52 (71 %)


FG-GAP 618..661 14/51 (27%) 0.073


32/51 (63%)


integrin 1121..1135 8115 (53%) 0.0048
A


( 14/15
93%)


Example
3.


The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 3A.
Table 3A. NOV3 Sequence Analysis
.._... _................W..............._..................................
....... .... .._......... .............. ...... ...... ........~.._... .
."u,~,:...................-.....................__......~...._.",~",,.~,
sa, CG50253-O1 SEQ.._~..N~.y.....s............................................
.....~ 1,882_.bp. ........... ...~.............
ORF Start .............~,~_ p .. _.. . . .....
..................................
Sequence : ATG at 243 ORF Sto : TAA at 1851
CTGACATGGACTGAAGGAGTAGAAAAGAAGGGAGCGGGAGGGGGCTCCGGGCGCCGCGCAGC
CTACTCCGGCCGCGCGCCTCGCCGCTGTCCTCCGGGAGCGGCAGCAGTAGCCCGGGCGGCGAGG
CTGGGGGTTCCTCGAGACTCTCAGAGGGGCGCCTCCCATCGGCGCCCACCACCCCAACCTGTTCCTC
CGCGCCACTGCGCTGCGCCCCAGGACCCGCTGCCCAACATGGATTTTCTCCTGGCGCTGGTGCTGGT
ACCTGCAGGCGGCCGCCGAGTTCGACGGGAGGTGGCCCAGGCAAATAGTGTCATCGA
TGTATCGGGCCAAACAAGTGCAAGTGTCA
CCTGGTTATGCTGGAAAAACCTGTAATCAAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTA
TATATGCTCATGCCG
CCATCCCCTGGCCTGCAGCTGGCTCCTGATGGGAGGACCTGTGTAG
AAAGGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGA
136



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ATCCCAAAAGTT
AATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTA
TAGCACCAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACA
TTTT
TCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCC
TATCTGACAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGC
a, CG50253-O1 SEQ m NO: 6 536 as BMW at 58650.8kD
iSequence _
~__
~LVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCKHG
CGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSM
IRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDL
GGKYQCHDIDECSLGQYQCSSFARCYNIRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPK
PPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELR
IAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGWIREKD
G
PIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKH
TLRGADIKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEER
NOV3b, 250095765 '~SEQ ll~ NO: 7 1765 by _
DNA Sequence O~' Start; at 2 ' ORF Stop: end of sequence
CACCGGATCCATGGATTTTCTCCTGGCGCTGGTGCTGGTATCCTCGCTCTACCTGCAGGCGGCCGCC
TGTCGTTATGGTGGGAGGAT
TGCAAACATGGTGAATGTATC
137



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CAACGGATATATGCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCAAACTGT
C
AGTATGGCTGTGATGTTGTTAAAGGACAAATACGGTGCCAGTGCCCATCCCCTGGCCTGCAGCTGGC
T
TGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTA
ATAACGTACGTGGGTCCTACAAGTGCAAATGTAAAGAAGGATACCAGGGTGATGGACTGA
TATCCCAAAAGTTATGATTGAACCTTCAGGTCCAATTCATGTACCAAAGGGAAATGGT
AAAGGGTGACACAGGAAATAATAATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCC
CACCTACAACCCCAGAAAGGCCAACCACCGGACTGACAACTATAGCACCAGCTGCCAGTACACCTCC
C
TAGAAAGAGGAGTCAGTGCAGACGATGAA
CAAAGGATGATCCAGGTGTTCTGGTACACAGTTGTAATTTTGACCATGGACTTTGTGGATGGATCAG
GAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCCAGCAGGTGGACAATATCTGACAGTGT
C
GGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCTACCTCTCGGCCGCCTCATGCATTCA
GGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGCACTCTGGCACACTCCAGGTGTTTGT
G
TCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAAAGGTGAAAAAAGGCGTGGTCACACT
TTGGATTAGATGATGTGAGCTTGAAAAAAGGCCACTGCTCTGAAGAACGCGTCGACGGC
250095765 ~SEQ m NO: 8 588 as ~MW at 64525.3kD
Sequence
VLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPFYVLRQ
VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYC
TGRASCPRF
CKCHKGFDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGL
IPKVMIEPSGPIHVPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPT
IPTPPPPPPLPTELRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFI
FEIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTV
VLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHGWRQT
ITLRGADIKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEERVDG
138



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
NOV3c, 250095742 SEQ m, NO: 9 _1627 bp_,.
____.._...... ~ p. _._..._._._.q......_..._._._.......___._.............
DNA Sequence ~ ORF Start: at 2 ORF Sto : end of se uence
__.. _..__.....____.._._._...._....._..._...~.._._._____ ._.-
_..__..____.__....-_..._._~..__._......_.._....._...__-...._w..~.~_H..._.
CACCGGATCCATGGATTTTCTCCTGGCGCTGGTGCTGGTATCCTCGCTCTACCTGCAGGCGGCCGCC
CTGAAGCCCCGGCCCTGTAAGCACAGGTGCATGAACACTTACGGCAG
TATATGCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTGACCT
CTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAAGGACAAATACGGTGCCAGTGCCCATCC
C
TGAATGTGCTACAGGAAGAGC
.TAGACGAATGCTCACTTGGTCAGTAT
TA
TGATTGAACCTTCAGGTCCAATTCATG
CAAAGGGAAATGGTACCATTTTAAAGGGTGACACAGGAAATAATAATTGGATTCCTGATGTTGGA
GTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTATCATTACCAACAGGCCTACTTCTAAGCC
ACACCAAAGCCAACACCAATTCCTACTCCACCACCACCACCACCCCTGCCAACAG
TCAGGG
TGCATTCAGG
G
GAGATTGGATTAGATGATGTGAGCTTGAAAAAAGGCCACTGCTCTGAAGAACGCGTCGACGGC
NOV3c,~250095742 ~~SEQ~m~.~N~O: 1542 aa~.~W at 591,62.3kD
Protein Seauence
VLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRC
IGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALT
IRCQCPSPGLHLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKG
GGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIH
139



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
PDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPT
PTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGWIR
SAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFV
ITLRGADIKSWFKGEKI2RGHTGEIGLDDVSLKKGHCSEERVDG
NOV3d, 250095779 SEQ m NO: 1,1. ...... ._1765..bp..._..___ .._._.._...._.~...
. __._........_......_....._......._..__._....
DNA Sequence O~' Start: at 2 ORF Stop: end of sequence
_...... _ _ . .. ~. ... ...~..~. . ..~.__. .___... . ~~__. _.~._...~.~_
.~___..._._.___
_CACCGGATCCATGGATTTTCTCCTGGCGCTGGTGCTGGTATCCTCGCTCTACCTGCAGGCGGCCGCC
G
T
GACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGTGTCAGCCTTTCTACGTCTTAAGGCAGA
AAGGTGCCAGCTCAAAGCTGTGTGCCAACCACGATGCAAACATGGTGAATGTATC
TGAGTG
CTGTGATGTTGTTAAAGGACAAATACGGTGCCAGTGCCCATCCCCTGGCCTGCACCTGGC
TGTTGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTA
TAAAGGCTTCGATCTCATGTATATT
TAGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGC
CGATGTTATAACGTACGTGGGTCCTACAAGTGCAAATGTAAAGAAGGATACCAGGGTGATGGACTGA
C
TATCCCAAAAGTTATGATTGAACCTTCAGGTCCAATTCATGTACCAAAGGGAAATGGT
CCATTTTAAAGGGTGACACAGGAAATAATAATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCC
C
TATATTCCTCCTATCATTACCAACAGGCCTACTTCTAAGCCAACAACAAGACCTACAC
AAAGCCAACACCAATTCCTACTCCACCACCACCACCACCCCTGCCAACAGAGCTCAGAACACCTCTA
C
CACCTACAACCCCAGAAAGGCCAACCACCGGACTGACAACTATAGCACCAGCTGCCAGTACACCTCC
C
ACAGACAGACCCTCAGAAACCCAGAGGAGATGTGTTCATTC
ATTTGAAATAGAAAGAGGAGTCAGTGCAGACGATGAA
CAAAGGATGATCCAGGTGTTCTGGTACACAGTTGTAATTTTGACCATGGACTTTGTGGATGGATCAG
C
GGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCTACCTCTCGGCCGCCTCATGCATTCA
G
GGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGCACTCTGGCACACTCCAGGTGTTTGT
G
TGGTGGCCATGGCTGGAGGCAAACAC
GATCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAAAGGTGAAAAAAGGCGTGGTCACACT
G
140



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
q SEQ mYNO: 124 -588yaa - ~MW at 64534.3kD
PrOot m Se500nce 79_.... .. ~_ .....___. _ "~ _..._....._ __._~_ .._~
.____...._ _ _._ ...
TGSMDFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPFYVLRQ
VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYC
SALTCSMANCQYGCDWKGQIRCQCPSPGLHLAPDGRTCVDVDECATGRASCPRF
QCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGL
T
CWIPKVMIEPSGPIHVPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPT
PTPPPPPPLPTELRTPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFI
FETFEIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTV
S
KAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHGWRQT
ITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEERVDG
OV3e, 250095794 SEQ DJ NO l3_~~1657 by
NA Sequence ORF Start: at 2 ~ ORF Stop: end of
CACCGGATCCGAGTTCGACGGGAGGTGGCCCAGGCAAATAGTGTCATCGATTGGCCTATGTCGTTAT
TCCTGGTTATGCTGGAA
TCAAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAAGCACAGGTGCATGAAC
TATATGCTCATGCCGGATGGTTCCTGCTCAAG
TGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAAGGACAAATACGGTGCC
CTGGCCTGCACCTGGCTCCTGATGGGAGGACCTGTGTAGATGTTGATGAATGTGCT
CAGGAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGAGCTACATCTGCAAGTG
T
c_'ATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGACATAGACGAATGCTCAC
TGT
CCAATTCATGTACCAAAGGGAAATGGTACCATTTTAAAGGGTGACACAGGAAATAATAATTGGATTC
TATTCCTCCTATCATTACCAACAGGCCT
C
CTGCCAACAGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCAACCACCGGACTGACAA
C
TTTGAAAT
141



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TGACCATGGACTTTGTGGATGGATCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGAC
C
CTCTCGGCCGCCTCATGCATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGC
ATGGTGGCCATGGCTGGAGGCAAACACAGATCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTT
C
V3e, 250095794 ~SEQ m NO: 14 552 as MW at 60397.3kD
~ein, Sequence . ...~. ~ _ _.; _. __ ,........._.._.._ .._._.__..___...~._..
.~._.~...._~~._.~.,~
EFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCKHGECIGPNKCKCHPGYAG
Q
CPSPGLHLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECS
L
GQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKGNGTILKGDTGNNNWI
P
DVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELRTPLPPTTPERPTTGLT
T
IAPAASTPPGGITVDNRVQTDPQKPRGDVFIPRQPSNDLFEIFEIERGVSADDEAKDDPGVLVH'SCN
F
DHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTG
L
HSGTLOVFVRKHGAHGAALWGRNGGHGWRQTQITLRGADIKSVVFKGEKRRGHTGEIGLDDVSLKKG
Sf, 250095734 '~SEQ m NO_ :_15__1717 by _
Sequence ORF Start: at 2 ORF Stop: end of
AGTTCGACGGGAGTAGGTGGCCCAGGCAAATAGTGTCATCGATTGGCCTATGTCGTTATGGTGGGAG
G
ATTGACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGTGTCAGCCTGTGTGCCAACCACGAT
G
eAAACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCATCCTGGTTATGCTGGAAAAACCTGT
ACTGTCTCAACGGATATATGCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTGA
AACTGTCAGTATGGCTGTGATGTTGTTAAAGGACAAATACGGTGCCAGTGCCCA
GCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGAGCTACATCTGCAAGTGTCATAAAG
G
CTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGACATAGACGAATGCTCACTTGGTCAG
T
ATCAGTGCAGCAGCTTTGCTCGATGTTATAACGTACGTGGGTCCTACAAGTGCAAATGTAAAGAAGG
142



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TGTACCAAAGGGAAATGGTACCATTTTAAAGGGTGACACAGGAAATAATAATTGGATTCCTGATGTT
G
GAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTATCATTACCAACAGGCCTACTTCTAA
G
CCAACAACAAGACCTACACCAAAGCCAACACCAATTCCTACTCCACCACCACCACCACCCCTGCCAA
C
AGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCAACCACCGGACTGACAACTATAGCA
C
CAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAGGGTACAGACAGACCCTCAGAAACC
C
AGAGGAGATGTGTTCATTCCACGGCAACCTTCAAATGACTTGTTTGAAATATTTGAAATAGAAAGAG
G
AGTCAGTGCAGACGATGAAGCAAAGGATGATCCAGGTGTTCTGGTACACAGTTGTAATTTTGACCAT
G
GACTTTGTGGATGGATCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCCAGCAGG
T
GGACAATATCTGACAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCTACCTC
T
CGGCCGCCTCATGCATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGCACTCT
G
GCACACTCCAGGTGTTTGTGAGAAAACACGGTGCCCACGGAGCAGCCCTGTGGGGAAGAAATGGTGG
C
CATGGCTGGAGGCAAACACAGATCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAAAGGTG
A
AAAAAGGCGTGGTCACACTGGGGAGATTGGATTAGATGATGTGAGCTTGAAAAAAGGCCACTGCTCT
G
AAGAACGCGTCGACGGC
NOV3f, 250095734 SEQ ID NO: 16 ~ 572 as MW at 62504.8kD~
Protein Sequence ~__~_
TGSMDFLLALVLVSSLYLQAAAEFDGSRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPR
C
KHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSAL
T
CSMANCQYGCDWKGQIRCQCPSPGLHLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHK
G
FDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPI
H
VPKGNGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLP
T
ELRTPLPPTTPERPTTGLTTIAPAASTPPGGITWNRVQTDPQKPRGDVFIPRQPSNDLFEIFEIER
G
VSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLP
L
GRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHGWRQTQITLRGADIKSWFKG
E
KRRGHTGEIGLDDVSLKKGHCSEERVDG
NOV3g, 250095811~ Y~ SEQ ID NO 17_N 6131bp
DNA Sequence p~ S_tart: at 2 - ORF Stop: end of sequence
_CACCGGATCCTGTCAGCCTGTGTGCCAACCACGATGCAAACATGGTGAATGTATCGGGCCAAACAAG
T
GCAAGTGTCATCCTGGTTATGCTGGAAAAACCTGTAATCAAGATCTAAATGAGTGTGGCCTGAAGCC
C
CGGCCCTGTAAGCACAGGTGCATGAACACTTACGGCAGCTACAAGTGCTACTGTCTCAACGGATATA
T
GCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGT
G
ATGTTGTTAAAGGACAAATACGGTGCCAGTGCCCATCCCCTGGCCTGCAGCTGGCTCCTGATGGGAG
G
143



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
ACCTGTGTAGATGTTGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCA
A
CACTTTTGGGAGCTACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATAT
C
AATGTCATGACATAGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAA
C
GTACGTGGGTCCTACAAGTGCAAATGTAAAGAAGGATACCAGGGTGATGGACTGACTTGTGTCGACG
G
C
NOV3g, 250095811 SEQ m NO: 18 204 as MW at 22351.4kD
Protein Sequence _
___.__.~ ~ __
TGSCQPVCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGY~
M
LMPDGSCSSALTCSMANCQYGCDVVKGQIRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRFRQCV
N
TFGSYICKCHKGFDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVD
G
NOV3h, 250095799 ~ SEQ m NO: 19_J 613 bp4 _
. .__ __ _.. - ~ __ _ _
DNA Sequence _ p~ Start: at 2 " ORF Stop:_end of sequence ~~
_CACCGGATCCTGTCAGCCTGTGTGCCAACCACGATGCAAACATGGTGAATGTATCGGGCCAAACAAGY
T
GCAAGTGTCATCCTGGTTATGCTGGAAAAACCTGTAATCAAGATCTAAATGAGTGTGGCCTGAAGCC
C
CGGCCCTGTAAGCACAGGTGCATGAACACTTACGGCAGCTACAAGTGCTACTGTCTCAACGGATATA
T
GCTCATGCCGGATGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGT
G
ATGTTGTTAAAGGACAAATACGGTGCCAGTGCCCATCCCCTGGCCTGCACCTGGCTCCTGATGGGAG
G
ACCTGTGTAGATGTTGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCA
A
CACTTTTGGGAGCTACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATAT
C
AATGTCATGACATAGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAA
C
GTACGTGGGTCCTACAAGTGCAAATGTAAAGAAGGATACCAGGGTGATGGACTGACTTGTGTCGACG
G
C
NOV3h, 250095799 SEQ m NO: 20 204 as MW at 22360.4kD
Protein Sequence
~, ~.~ ~__ ...;,~
_- ~, __.._ ~_._
TGSCQPVCQPRCHIiGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGY
M
LMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLHLAPDGRTCVDVDECATGRASCPRFRQCV
N
TFGSYICKCHKGFDLMYIGGKYQCHDIDECSLGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVD
G
NOV3i, SNP13377609 of SEQ m NO: 21 _ 1882 by
CG50253-O1, DNA Sequence OgE Start: ATG at 243 O_RF_Stop~:4_TAA at_1851
_ : SNP Pos: 63 SNP Ch_ange:_G t_o A_
GGACACTGACATGGACTGAAGGAGTAGAAAAGAAGGGAGCGGGAGGGGGCTCCGGGCGCCGCACAGCA
GACCTACTCCGGCCGCGCGCCTCGCCGCTGTCCTCCGGGAGCGGCAGCAGTAGCCCGGGCGGCGAGGG
CTGGGGGTTCCTCGAGACTCTCAGAGGGGCGCCTCCCATCGGCGCCCACCACCCCAACCTGTTCCTCG
CGCGCCACTGCGCTGCGCCCCAGGACCCGCTGCCCAACATGGATTTTCTCCTGGCGCTGGTGCTGGTA
TCCTCGCTCTACCTGCAGGCGGCCGCCGAGTTCGACGGGAGGTGGCCCAGGCAAATAGTGTCATCGAT
TGGCCTATGTCGTTATGGTGGGAGGATTGACTGCTGCTGGGGCTGGGCTCGC'~AC~Trmmrrr~r_n rar_m
144



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
GGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCAT
TCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAA
AGTGCTACTGTCTCAACGGATATATGCTCATGCCGG
ATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAA
TACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGAC
AGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAACATACGTGGGTC
AATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTAT
CAACAGGCCTACTTCTAAGCCAACAACAAGACCTACACCAAAGCCAACACCAATTCCTACTC
CACCACCACCCCTGCCAACAGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCA
GGACTGACAACTATAGCACCAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAG
TATCTGACAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCT
CCTCATGCATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGC
TCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAA
AGGTGAAAP.AP.GGCGTGGTCP.CACTGGGGAGATTGGATTAGATGATGTGAGCTTGAAAAAAGGCCACT
GCTCTGAAGAACGCTAACAACTCCAGAACTAACAATGAACTCCTAA
)V3i, SNP13377609 of SEQ m NO: 22.536 as MW a_t _58_650.8kD
......... ... .
X50253-O1, Protein Sequence ~ SNP Change: no change
FLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCKHGE
GPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMA
QYGCDVVKGQIRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLM
GGKYQCHDIDECSLGQYQCSSFARCYNIRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKG
TILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELRT
PPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGWIREKDN
HWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHG
GAALWGRNGGHGWRQTQITLRGADIKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEER
~j, SNP13373929 of ~SEQ ID NO: 23 1882,bp..
253-O1, DNA Sequence ~O~~Start: ATG at 243 ORF Ston: TAA at 1851
Pos: 226 SNP Change: A to G
TGGACTGAAGGAGTAGAAAAGAAGGGAGCGGGAGGGGGCTCCGGGCGCCGCGCAGCA
TTTTCTCCTGGCGCTGGTGCTGGTA
CGAGTTCGACGGGAGGTGGCCCAGGCAAATAGTGTCATCGAT
TTGACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGT
AAACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCAT
TCAAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAA
CTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAA
CCCATCCCCTGGCCTGCAGCTGGCTCCTGATGGGAGGACCTGTGTAGA
GAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGA
AAAGGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGAC
TCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAACATACGTGGGTC
CTCCGAAGACACCATATATTCCTCCTAT
ACACCAAAGCCAACACCAATTCCTACTC
ATAGCACCAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAG
TGGATCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCCA
145



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
GATTGGATTAGATGATGTGAGCTTGAAAAAAGGCCACT
AACAATGAACTCCTAA
NOV3j, SNP13373929 of SEQ )D NO: 24536 aa. MW at.58650.8kD
CG50253-O1, Protein Sequence ' SNP Change: no change
MDFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCKHGE
CTGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMA
NCQYGCDWKGQIRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLM
YIGGKYQCHDIDECSLGQYQCSSFARCYNTRGSYKCKCKEGYQGDGLTCWTPKVMIEPSGPIHVPKG
NGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELRT
ERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGWIREKDN
RDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHG
GRNGGHGWRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEER
SNP13380272 of ~SEQ m_ _N_O_: 25 -" 1882 bp_ _ _ _
3-O1, DNA Sequence ; Ogp Start: ATG at 243 ORF Ston: TAA at 1851
Pos: 719 ~f SNP Change: G to C
GGACACTGACATGGACTGAAGGAGTAGAAAAGAAGGGAGCGGGAGGGGGCTCCGGGCGCCGCGCAGCA
GACCTACTCCGGCCGCGCGCCTCGCCGCTGTCCTCCGGGAGCGGCAGCAGTAGCCCGGGCGGCGAGGG
CTGGGGGTTCCTCGAGACTCTCAGAGGGGCGCCTCCCATCGGCGCCCACCACCCCAACCTGTTCCTCG
CGCGCCACTGCGCTGCGCCCCAGGACCCGCTGCCCAACATGGATTTTCTCCTGGCGCTGGTGCTGGTA
TCCTCGCTCTACCTGCAGGCGGCCGCCGAGTTCGACGGGAGGTGGCCCAGGCAAATAGTGTCATCGAT
TGGCCTATGTCGTTATGGTGGGAGGATTGACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGT
GTCAGCCTGTGTGCCAACCACGATGCAAACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCAT
CCTGGTTATGCTGGAAAAACCTGTAATCAAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAA
GCACAGGTGCATGAACACTTACGGCAGCTACAAGTGCTACTGTCTCAACGGATATATGCTCATGCCGG
ATGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAA
GGACAAATACGGTGCCAGTGCCCATCCCCTGGCCTGCA_CCTGGCTCCTGATGGGAGGACCTGTGTAGA
TGTTGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGA
GCTACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGAC
ATAGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAACATACGTGGGTC
TTTTAAAGGGTGACACAGGA
AATAATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTAT
CACCACCACCACCACCCCTGCCAACAGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCA
ACCACCGGACTGACAACTATAGCACCAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAG
ACCCAGAGGAGATGTGTTCAGTGTTCTGGTACACAGTTGTAATTTTG
TCAGGGAGAAAGACAATGACTTGCACTGGGAACCAATCAGGGACCCA
TCTGACAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCT
TCATGCATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGC
TTAGATGATGTGAGCTTGAAAAAAGGCCACT
fOV3k, SNP13380272 of ,SEQ )D NO: 26 536 as MW at 58659.8kD
'G50253-O1, Protein Sequence SNp Pos: 159 _ SNP Change: Gln to His
DFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCKHGE
IGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMA
CQYGCDWKGQIRCQCPSPGL_HLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLM
IGGKYQCHDIDECSLGQYQCSSFARCYNIRGSYKCKCKEGYQGDGLTCWIPKVMTEPSGPIHVPKG
GTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELRT
LPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGWIREKDN
LHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHG
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TLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEER
OV31, SNP13379745 of ~SEQ ID NO_ .:,_21.882 bp. _
650253-Ol, DNA Sequence 'O~~Start: ATG at 243 ORF Stop: TAAYat 1851
SNP Pos: 750 SNP Change: G to A
~GACCTACTCCGGCCGCGCGCCTCGCCGCTGTCCTCCGGGAGCGGCAGCAGTAGCCCGGGCGGCGAGGG
G'1'c:AGC:C:'1 G'1 G TGCCAACCACGATGCAAACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCAT
CCTGGTTATGCTGGAAAAACCTGTAATCAAGATCTAAATGAGTGTGGCCTGAAGCCCCGGCCCTGTAA
GCACAGGTGCATGAACACTTACGGCAGCTACAAGTGCTACTGTCTCAACGGATATATGCTCATGCCGG
ATGGTTCCTGCTCAAGTGCCCTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAA
GGACAAATACGGTGCCAGTGCCCATCCCCTGGCCTGCAGCTGGCTCCTGATGGGAGGACCTGTGTAGA
TA_TTGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGA
GCTACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGAC
ATAGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAACATACGTGGGTC
CTACAAGTGCAAATGTAAAGAAGGATACCAGGGTGATGGACTGACTTGTGTGTATATCCCAAAAGTTA
TGATTGAACCTTCAGGTCCAATTCATGTACCAAAGGGAAATGGTACCATTTTAAAGGGTGACACAGGA
AATAATAATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTAT
CATTACCAACAGGCCTACTTCTAAGCCAACAACAAGACCTACACCAAAGCCAACACCAATTCCTACTC
CACCACCACCACCACCCCTGCCAACAGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCA
TGTGTTCAGTGTTCTGGTACACAGTTGTAATTTTG
GGTGGCCATGGCTGGAGGCAAACACAGATCACCTTGCGAGGGGCTGACATCAAGAGCGTCGTCTTCAA
AGGTGAAAAAAGGCGTGGTCACACTGGGGAGATTGGATTAGATGATGTGAGCTTGAAAAAAGGCCACT
GCTCTGAAGAACGCTAACAACTCCAGAACTAACAATGAACTCCTAA
NOV31, SNP13379745 of ~SEQ ID NO: 28 536 as ,M_W_a_t 58_664.8kD _
CG50253-Ol, Protein Sequence ~ SNP Pos: 170 SNP Change: Val to Ile
MDFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCKHGE
CIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMA
NCQYGCDVVKGQIRCQCPSPGLQLAPDGRTCVD_IDECATGRASCPRFRQCVNTFGSYICKCHKGFDLM
YIGGKYQCHDIDECSLGQYQCSSFARCYNIRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKG
NGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELRT
PLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGWIREKDN
DLHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHG
AHGAALWGRNGGHGWRQTQITLRGADIKSVVFKGEKRRGHTGEIGLDDVSLKKGHCSEER
n, SNP13373930 of SEQ ID NO: 29 1882 by
53-O1, DNA Sequence ~Ogp Start: ATG at 243 ORF Stop: TAA at 1851
Pos: 942 SNP Change: A to G
TGTCGTTATGGTGGGAGGATTGACTGCTGCTGGGGCTGGGCTCGCCAGTCTTGGGGACAGT
TGTGTGCCAACCACGATGCAAACATGGTGAATGTATCGGGCCAAACAAGTGCAAGTGTCAT
TGAACACTTACGGCAGCTACAAGTGCTACTGTCTCAACGGATATATGCTCATGCCGG
TCAAGTGCCCTGACCTGCTCCATGGCAAACTGTCAGTATGGCTGTGATGTTGTTAAA
GTGCCAGTGCCCATCCCCTGGCCTGCAGCTGGCTCCTGATGGGAGGACCTGTGTAGA
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TGTTGATGAATGTGCTACAGGAAGAGCCTCCTGCCCTAGATTTAGGCAATGTGTCAACACTTTTGGGA
GCTACATCTGCAAGTGTCATAAAGGCTTCGATCTCATGTATATTGGAGGCAAATATCAATGTCATGAC
ATAGACGAATGCTCACTTGGTCAGTATCAGTGCAGCAGCTTTGCTCGATGTTATAACGTACGTGGGTC
TGATTGAACCTTCAGGTCCAATTCATGTACCAAAGGGAAATGGTACCATTTTAAAGGGTGACACAGGA
AATAATAATTGGATTCCTGATGTTGGAAGTACTTGGTGGCCTCCGAAGACACCATATATTCCTCCTAT
CATTACCAACAGGCCTACTTCTAAGCCAACAACAAGACCTACACCAAAGCCAACACCAATTCCTACTC
CACCACCACCACCACCCCTGCCAACAGAGCTCAGAACACCTCTACCACCTACAACCCCAGAAAGGCCA
ACCACCGGACTGACAACTATAGCACCAGCTGCCAGTACACCTCCAGGAGGGATTACAGTTGACAACAG
ACACAGTTGTAATTTTG
GACAGTGTCGGCAGCCAAAGCCCCAGGGGGAAAAGCTGCACGCTTGGTGCT
TGCATTCAGGGGACCTGTGCCTGTCATTCAGGCACAAGGTGACGGGGCTGC
TCAAGAGCGTCGTCTTCAA
AACAACTCCAGAACTAACAATGAACTCCTAA
NOV3m SNP13373930 of SEQ ID NQ: 30536 as ~MW at 58636.8kD
a q Pos 234......S~~Change.......~......._._.....~.~-...-~....x.
CG50253 O1 Protein Se uence . : Ile to Val
MDFLLALVLVSSLYLQAAAEFDGRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQCQPVCQPRCKHGE
CIGPNKCKCHPGYAGKTCNQDLNECGLKPRPCKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMA
NCQYGCDWKGQIRCQCPSPGLQLAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLM
YIGGKYQCHDIDECSLGQYQCSSFARCYNV_RGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKG
NGTILKGDTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELRT
PLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFSVLVHSCNFDHGLCGWIREKDN
DLHWEPIRDPAGGQYLTVSAAKAPGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHG
AHGAALWGRNGGHGWRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEER
A ClustalW comparison of the above protein sequences yields the following
sequence
alignment shown in Table 3B.
Table 3B. Comparison of the l~TOV3 protein sequences.
NOV3a ---MDFLLALVLVSSLYLQAAAEFDG-RWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQC
NOV3b TGSMDFLLALVLVSSLYLQAAAEFDG-RWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQC
NOV3c TGSMDFLLALVLVSSLYLQAAAEFDG-RWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQC
NOV3d TGSMDFLLALVLVSSLYLQAAAEFDG-RWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQC
NOV3e -------------------TGSEFDG-RWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQC
NOV3f TGSMDFLLALVLVSSLYLQAAAEFDGSRWPRQIVSSIGLCRYGGRIDCCWGWARQSWGQC
NOV3g ________________________________________________________TGSC
NOV3h ________________________________________________________TGSC
NOV3a QP-----------------VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3b QPFYVLRQRIARIRCQLKAVCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3c QP-----------------VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3d QPFYVLRQRIARIRCQLKAVCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3e QP-----------------VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3f QP-----------------VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3g QP-----------------VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3h QP-----------------VCQPRCKHGECIGPNKCKCHPGYAGKTCNQDLNECGLKPRP
NOV3a CKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLQ
NOV3b CKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLQ
NOV3c CKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLH
NOV3d CKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLH
NOV3e CKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDVVKGQIRCQCPSPGLH
NOV3f CKFiRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLH
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NOV3g CKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDWKGQIRCQCPSPGLQ
NOV3h CKHRCMNTYGSYKCYCLNGYMLMPDGSCSSALTCSMANCQYGCDVVKGQIRCQCPSPGLH
NOV3a LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECS
NOV3b LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCFH)IDECS
NOV3c LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECS
NOV3d LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECS
NOV3e LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECS
NOV3f LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECS
NOV3g LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHDIDECS
NOV3h LAPDGRTCVDVDECATGRASCPRFRQCVNTFGSYICKCHKGFDLMYIGGKYQCHI~IDECS''
NOV3a LGQYQCSSFARCYNIRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKGNGTILKG
NOV3b LGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKGNGTILKG~
NOV3c LGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCWIPKVMIEPSGPIHVPKGNGTILKG
NOV3d LGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCWIPKVMIEPSGPIHVPKGNGTILKG
NOV3e LGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCWIPKVMIEPSGPIHVPKGNGTILKG
NOV3f LGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVYIPKVMIEPSGPIHVPKGNGTTLKG
NOV3g LGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVDG-----------------_____
NOV3h LGQYQCSSFARCYNVRGSYKCKCKEGYQGDGLTCVDG-----------------_____
NOV3a DTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELR
NOV3b DTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELR
NOV3c DTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELR
NOV3d DTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELR
NOV3e DTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELR
NOV3f DTGNNNWIPDVGSTWWPPKTPYIPPIITNRPTSKPTTRPTPKPTPIPTPPPPPPLPTELR
NOV3g ____________________________________________________________
NOV3h ____________________________________________________________
NOV3a TPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFS-----------
NOV3b TPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFIPRQPSNDLFEIF
NOV3c TPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFS-----------
NOV3d TPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFIPRQPSNDLFEIF
NOV3e TPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFIPRQPSNDLFEIF
NOV3f TPLPPTTPERPTTGLTTIAPAASTPPGGITVDNRVQTDPQKPRGDVFIPRQPSNDLFEIF
NOV3g ____________________________________________________________
NOV3h ____________________________________________________________
NOV3a -----------------VLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAK
NOV3b EIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAK
NOV3c ----------------_~,WSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAK
NOV3d EIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAK
NOV3e EIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAK
NOV3f EIERGVSADDEAKDDPGVLVHSCNFDHGLCGWIREKDNDLHWEPIRDPAGGQYLTVSAAK
~NOV3g ____________________________________________________________
',NOV3h ____________________________________________________________
NOV3a APGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHG
NOV3b APGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHG
NOV3c APGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRHI3GAHGAALWGRNGGHG
NOV3d APGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHG
NOV3e APGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRKHGAHGAALWGRNGGHG
NOV3f APGGKAARLVLPLGRLMHSGDLCLSFRHKVTGLHSGTLQVFVRIQiGAHGAALWGRNGGHG
NOV3g ____________________________________________________________
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NOV3h
~NOV3a WRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEER---
NOV3b WRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEERVDG
NOV3C WRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEERVDG
NOV3d WRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEERVDG
NOV3e WRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEERVDG
NOV3f WRQTQITLRGADIKSWFKGEKRRGHTGEIGLDDVSLKKGHCSEERVDG
NOV3g _________________________________________________
NOV3h _________________________________________________
NOV3a(SEQ IDNO: 6)


NOV3b(SEQ IDNO: 8)


NOV3c(SEQ IDNO: 10)


NOV3d(SEQ IDN0: 12)


NOV3e(SEQ IDNO: 14)


NOV3f(SEQ IDN0: 16)


NOV3g(SEQ IDNO: 18)


NOV3h(SEQ IDNO: 20)


Further analysis of the NOV3a protein yielded the following properties shown
in Table 3C.
Table 3C. Protein Sequence Properties NOV3a
SignalP analysis: Cleavage site between residues 20 and 21~
PSORT II analysis:
PSG: a new signal peptide prediction method
N-region: length 2; pos.chg 0; neg.chg 1
H-region: length 17; peak value 0.00
PSG score: -4.40
GvH: von Heijne's method for signal seq. recognition
GvH score (threshold: -2.1): -0.34
possible cleavage site: between 17 and 18
» > Seems to have no N-terminal signal peptide
ALOM: Klein et al's method for TM region allocation
Init position for calculation: 1
Tentative number of TMS(s) for the threshold 0.5: 1
Number of TMS(s) for threshold 0.5: 1
INTEGRAL Likelihood = -4.19 Transmembrane 3 - 19
PERIPHERAL Likelihood = 5.67 (at 439)
ALOM score: -4.19 (number of TMSs: 1)
MTOP: Prediction of membrane topology (Hartmann et al.)
Center position for calculation: 10
Charge difference: 0.0 C( 0.0) - N( 0.0)
N >= C: N-terminal side will be inside
» > membrane topology: type 2 (cytoplasmic tail 1 to 3)
MITDISC: discrimination of mitochondrial targeting seq
R content: 0 Hyd Moment(75): 4.41
Hyd Moment(95): 7.23 G content: 0 _........
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D/E content: 2 S/T content: 2
Score: -6.55
Gavel: prediction of cleavage sites for mitochondria) preseq
cleavage site motif not found
NUCDISC: discrimination of nuclear localization signals
pat4: none
pat7: none
bipartite: none
content of basic residues: 11.9%
NLS Score: -0.47
KDEL: ER retention motif in the C-terminus: none
ER Membrane Retention Signals: none
SKG: peroxisomal targeting signal in the C-terminus: none
PTS2: 2nd peroxisomal targeting signal: none
VAC: possible vacuolar targeting motif: none
RNA-binding motif: none
Actinin-type actin-binding motif:
type 1: none
type 2: none
NMYR: N-myristoylation pattern : none
Prenylation motif: none
memYQRL: transport motif from cell surface to Golgi: none
Tyrosines in the tail: none
Dileucine motif in the tail: none
checking 63 PROSITE DNA binding motifs: none
checking 71 PROSITE ribosomal protein motifs: none
checking 33 PROSITE prokaryotic DNA binding motifs: none
NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination
Prediction: nuclear
Reliability: 89
COIL: Lupas's algorithm to detect coiled-coil regions
total: 0 residues
Final Results (k = 9/23):
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43.5 %: nuclear
26.1 %: mitochondria!
8.7 %: cytoplasmic
4.3 %: plasma membrane
4.3 %: vesicles of secretory system
4.3 %: extracellular, including cell wall
4.3 %: endoplasmic reticulum
4.3 %: peroxisomal
» prediction for CG50253-01 is nuc (k=23)
A search
of the
NOV3a protein
against
the Geneseq
database,
a proprietary
database
that


contains publication,
sequences yielded
published several
in patents homologous
and patent


proteins
shown in
Table 3D.


Table 3D.
Geneseq
Results
for NOV3a


NOV3a Identities/


Geneseq Protein/OrganismlLength Residues/SimilaritiesExpect
[Patent for


Identifier #, Date] Match the Matched Value
'


Residues Region


ABG70290 Human novel polypeptide 1..536 536/536 (100%)0.0
#6 -


Homo Sapiens, 536 aa. 1..536 536/536 (100%)


[W0200257452-A2, 25-JUL-2002]


ABG69659 Human secreted protein 1..536 534/566 (94%)0.0
. SCEP-39 -


Homo Sapiens, 566 aa. 1..566 535/566 (94%)


[WO200248337-A2, 20-JUN-2002]


AAB70547 Human PR017 protein sequence1..536 532/582 (91%)0.0


SEQ ID N0:34 - Homo sapiens,1..582 533/582 (91%)


582 aa. [W0200110902-A2,
15-


FEB-2001 ]


AAB70549 Clone 16467945Ø85-5261.D20..536 512/546 (93%)0.0


protein sequence SEQ 1..546 514/546 (93%)
ID N0:82 -


Homo Sapiens, 546 aa.


[WO200110902-A2, 15-FEB-2001]


ABU58425 3 Human PRO polypeptide 1..505 503/505 (99%)0.0
#26 -


Homo Sapiens, 509 aa. 1..505 ~ 504/505
(99%)


[US2003027272-Al, 06-FEB-2003]


In a BLAST the NOV3a
search protein
of public was found
sequence to have
databases,


homology
to the
proteins
shown in
the BLASTP
data in
Table 3E.


Table 3E. Results
Public for NOV3a
BLASTP


NOV3a Identities/


Protein
Residues/SimilaritiesExpect
for


Accession Match the Matched Value
Protein/Organism/Length


Number Residues Portion ~


CAC33425 0.0


152



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WOOI10902 - Homo 1..582 533/582 (91%)
sapiens


(Human), 582 aa.


Q91V88 POEM (NEPHRONECTIN 470/562 (83%) 0.0
short 1..532


isoform) - Mus musculus1..561 497/562 (87%)


(Mouse), 561 aa.


Q91ZD3 Nephronectin long 470/579 (81%) 0.0
isoform - Mus 1..532


musculus (Mouse), 1..578 497/579 (85%)
578 aa.


Q91XL5 Nephronectin - Mus 1..532 470/593 (79%) 0.0
musculus


(Mouse), 592 aa. 1..592 497/593 (83%)


Q923T5 Nephronectin - Mus 1..532 470/610 (77%) 0.0
musculus


(Mouse), 609 aa. 1..609 497/610 (81%)


PFam analysis
predicts
that the
NOV3a
protein
contains
the domains
shown
in the
Table


3F.


Table 3F. Domain
Analysis of NOV3a


Identities/


Pfam DomainNOV3a Match Region SimilaritiesExpect Value


for the Matched Region


C_tripleX 54..71 7/18 (39%) 0.24


15/18 (83%)


EGF 60..86 13/47 (28%)0.13


23/47 (49%)


EGF 93..127 15/47 (32%)0.51


27/47 (57%)


EGF 133..167 10/47 (21%)0.035


24/47 (51
%)


EGF 218..253 12/47 (26%)0.00011


28/47 (60%)


MAM 393..534 48/183 (26%)8.6e-27


98/183 (54%)


Example 4.
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide
sequences are
shown in Table 4A.
Table 4A. NOV4 Seauence An
4a, CG50377-04 ~SEQ ID NO: 31 -_10655 by _
Sequence ORF Start: at 1 ~ ORF Ston: TAG at 10639
CTACCCCAATTACGCCAACTGCACGTGGACCATCACCGCGGAAGAGCAGCACAGAATCCAGCTTGTG
CTTTGCCCTGGAAGAGGACTTTGATGTCCTGTCGGTGTTTGATGGTCCACCCCAGCCAGA
153



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TTGTTAGTGCAGCCACCACCCTCT
TGCCTGTGGT
TGGACCATCCTGGCTGAGCTGGGGGACACCATCGCCCTGGTGTTTATTGACTTCCAGC
GGAGGATGGTTACGACTTTCTGGAAGTCACTGGGACAGAAGGCTCCTCCCTCTGGTTCACCGGAGCC
A
TCGGATGGCAACCA
TTCAGTGCCCAATACCAAGTCAAGAAGCAAATTGAGTTGAAGTCTCGAGGTGTGA
ATAATATGTGTCCAGACCCTGGCATACCCGAAAGGGGCAAAAGACTAGGCTCGGATTTCAGGTTAGG
A
TCCAGCGTCCAGTTCACCTGCAACGAGGGCTATGACCTGCAAGGGTCCAAGCGGATCACCTGTATGA
A
TGTGTGATGCC
TCATCACCTCCCCCAATTTCCCCATTCAGTATGACAACAATGCACA
TT
ATGACACCCTGACGGTCGGTGATGGTGGTCAGGATGGGGACCAGAAGACAGTTCTC
TCGGTCCCGGATCTCATTGTCAGCACCAATCATCAAATGTGGCTCCTCTT
TGGCAGTGGCAGTTCCCTGGGATTCAAGGCTTCTTATGAAGAGATCGAGCAGGGCAGTT
CGGTGACCCTGGCATACCTGCATATGGCCGGAGGGAAGGCTCCCGGTTTCGCCACGGTGACACACTC
A
TGGCAACCACCTCCACTGTGTCTGGCTCATCCTGGCCAGG
CTGAGAGCCGCATCCACCTGGCCTTCAACGACATTGACGTGGAGCCTCAGTTTGATTTCCTGGTCAT
r.
TGAAGGCTTCCTTGGGACTC
TCACCTGCGTCCTGAAGGAGGGCAGCGTGGTCTGGAACAGCGCTGTGCTGCGG
154



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CGCCCTTGATCGGGGTTTACCACGGGACCCAGGTTCCCCAGTTCCTCATCAGCACCAGCAACTACCT
CAGCTCCGCTATGAGGCTATAA
AAATGGACAGCGTCATGGGAATGACTTC
AAGTGACGGGGAGCCTCTGGA
CAGGTTTCCCTGACTTCTACCCCAACAACTTGAACTGCACC
GGATTATCGAAACATCTCATGGCAAGGGTGTGTTCTTCACTTTCCACACCTTCCACCTGGAAAGTGG
ATGAAGGATTCAACATCACCTTCTCAGAGTACGACTTGGAGCCCTGTGAGGAGCC
CTTCCCCGGGTACCGTCTGGAGGGCACCGCCCGCATCACGTGCCTGGGGGGCAGACGGCGCCTGTGG
CAAGGTGTGTTGCTGAGTGTGGGAATTCAGTCACAGGCACTCAGGGTACTTTGCT
CCGTTTGCTGGGAGTTTTTAGCCATTCTGAGATGATGGGGGTGACTTTGAA
GCACTTTTCCAGTTTTGAACTCATCAAATGTGAGGACCCAGGAACCCCCAAGTTTGGCTACAAGGTT
ACAGCCTGCGGGG
C
CGAGTGTGGAGGGACAGTGAGAGGAGAGGTGTCGGGGCAGGTGCTGTCACCCGGGTATCCAGCTCCC
TCTGGACCATCGAAGCAGAGGCCGGCTGCACCATTGGGCTACACTT
TCTGCTGAAGGAGCTGAGTGGCCCGGCCCTGCCCAAGGACCTGCATAGCACCTTCAACTCGGTCGTC
CTGCAATGACCCTGGGATCCCGCAGAATGGGAGTCGGAGTGGTGACAGTTGGGAAGCCGGCG
TCAGCTGTGTGAAG
CTGGCAGCCCAGCCCGCCAACATGCATCGCTCCCTGCGGGGGAGACCTGAC
TCTGGAGTCATCCTCTCACCAAATTACCCAGAACCCTACCCGCCAGGCAAGGAGTGTGACT
155



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
ACGTCATCGCCCTGGTATTGTTCCCCAGCTTTAACCTGGAGCCT
GCTATGACTTCCTCCATATCTACGACGGACGGGACTCTCTCAGCCCTCTCATAGGAAGCTTCTATGG
TTGAAAGCAGCAGCAACAGCCTCTTCCTCGCCTTCCGCAGCGATGCAT
CCATCAAGAACGGCACACGGGTGGGGTCCGACCTGAAGCTGGGCTCCTCCGTCACCTACTACTGCCA
TGTGGGTTCGGACGGAGTGGTC
CAACTACCCCCAGAACTACACCAGTGGACAGATCTGCTTGTATTTTGTTACTGTGCCCAA
GACTATGGTGTGGTGTTTGGCCAGTTCGCCTTCTTTCACACGGCCCTCAACGACGTGGTGGAGGTTC
CACTTTGTCTACCAAGCGGTTCCTCGAACCAGCGCCACGCAGTGCAGCTCTGTGCCGGAACCCCGCT
AT
CGCCAGAGATCGAGTGCCTCCCTGTGCCTGGGGCCTTGGCCCAATGGAATGTCTC
CTCACAGAGCGCAGGGGCACCATCCTGTCCCCTG
CTTCCCAGAGCCGTACCTCAACAGCCTCAACTGTGTGTGGAAGATCGTGGTCCCCGAAGGCGCTGGC
TCCAGATCCAAGTTGTCAGTTTTGTGACAGAGCAGAACTGGGACTCGCTGGAAGTATTTGATGGTGC
AACCATGCTGGGGAGTTTCTCAGGAACAACCGTGCCTGCCCTTCTGAACAGCACCT
TTTCTACTCAGATATCAGCGTATCTGCAGCTGGCTTCCACTTGGAGTAC
TGCCCTCCAGGGCCACGCCCACA
TGGAACTACCCTCCTCCACTCTGTATTGCACAGTGTGGG
TCCTGAGCCCCGGCTTCCCAGGCAACTACCCCAGTAACAT
CGAGCCCAACCACGACTACATAGAAATCCGGAATGGCCCCTATGAGACCAGCCGCATGATGGGAAGA
TGGCATTGTGAGGGGAGCTGGCTACAACGTGGGACAATCAGTGACCTTC
TGGGACCACCCCCTGCCCAAGTGTGAAGTCCCTTGTGGCGGGAACATCACTTCTTCCAACGGCACTG
156



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TCCTCCCCAACGCCGAAGTCGT
TGAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCTCCCTGGCTTTACCTTAG
TATGT
TGAGCTTCTGACAGACTCCACAGGCGTGATCCTGAGCCAGAGCTACCC
TCCCCAGTTCCAGACCTGCTCTTGGCTGGTGAGAGTGGAGCCCGACTATAACATCTCCC
CACAGTGGAGTACTTCCTCAGCGAGAAGCAATATGATGAGTTTGAGATTTTTGATGGTCCATCAGGA
C
ACAATCGGAAGGGCTTCAAGATCCGCTATTCAG
CCCTTACTGCAGCCTGCCCAGGGCTCCACTCCATGGCTTCATCCTAGGCCAGACCAGCACCCAGCCC
r~_
TCCACTTTGGCTGCAACGCCGGCTACCGCCTGGTGGGACACAGCATGGCCATCTGTAC
CAGGGCTACCACCTGTGGAGCGAAGCCATCCCTCTCTGTCAAGCTCTTTCCTGTGGGC
TGGTGTTTGGCAAGGAGTACACAGTGGGAACCAAGGCCATGTAC
TGGAGGCTTATCTTTGAGACACAGTATCAGTTCCAGGCCCAGCTGATGCTCATC
GTGACCCTGGCTACTACTATACTGGCCAAAGGGTCATCCGCTGTCAGGCCAATGGCAAATGGAGCCT
C
CCGATTCCCCCCAATGGCCACC
ACGGGGCAACAGCCATCTTCTCCTGCAATTCCGGATACACACTGGTG
TGTGGTGTACCAATGCAATGCTGGCTTCCGCCTGATCGGCATGTCTGTGCGCATCTGCCAGCAGGAT
C
TA
AGGTCCCAATGCCTGGCCAGCGGGCAATGGAGTGACATGCTGCCCACCTGC
CACAGGTTCAGCTTCGGCACCACTGTGTCTTACCGGTGCAACCACGGCTTCTACCTCCTGGGCACCC
r~
157



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CGCCCCCAGTGTCTCCTGGTGTCCTGT
TGTCTGGAGACAGTTATACTGTGGGAGCAGTGGTGCG
TACTACGCCACAGGCCTGCTCAGCCGTCA
CTG
CCTGGCTATATGATGGAGTCACATAGAGTATCTGTGCTGAGCTGCACCAAGGACCGGACATGGAA
TGGGAAGGTGG
GGGGTCTGACTTCATGTGGGGCTCAAGTGTGACTTATGCCTGCCTGGAGGGGTACCAGCTCTCCCTG
T
CAGTTTGGGATACAGAACAATTCTCAGGGCTACCAGGTTGGAAGCACAGTCCTCTTCCGTTGTCAAA
ACCTGCTTCAGGGCTCCACCACCAGGACCTGCCTCCCAAACCTGACCTGGAGTGGAACCCCA
CTCAAGGGTGGCTCCG
GCACCGCACCTGCAAGGCGGATGGCAGCTGGACAGGCAAGCCGCCCATCTGCCTGGAGGTCCGGCCC
TGTTTT
TTCCCTGTGGAAAGGGGCCTATGAATACCAGGGGAAGAAGCAGCCAGCCATGCTCAGAG
GACTGGCTTCCAAGTTGCCAACAGCAAGGTCAATGCCACCATGATCGACCACAGTGGCGTGGAGCTG
CTACTCCAGGTGTACCAGATTACAGGGCC
TGAATAAGTTCAAAGATGATCACTGGGCTTTAGATGGCCATGTCTCGTCAGAGT
C
CTCCGGAGCCACCTTCATCTACCAAGGCTCTGTCAAGGGCCAAGGCTTTGGGCAGTTCGGCTTTCAA
TCCTGGTGCCTTTCATCGCCCTCATTATTGCGGGCTTCGTGCTCTATCTCT
1$g



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TTTGAGAACCCAATGTACGACCGCAACATCCAGCCCACAGACATCATGGCCAGCGAGGCGGA
4a, CG50377-04 ~SEQ m NO: 32 3546 as MW at 385864.8kD
in Sequence
TAEEQHRTQLVFQSFALEEDFDVLSVFDGPPQP
ATIVSAATTLSLRLISDYAVSAQGFHATYEVLPSHTCGNPGRLPNGIQQGSTFNL
CNLGFFLEGHAVLTCHAGSENSATWDFPLPSCRADDACGGTLRGQSGIISSPHFPSEYHNN
IALVFIDFQLEDGYDFLEVTGTEGSSLWFTGASLPAPVISSKNWLRLHFTSDGN
IELKSRGVKLMPSKDNSQKTSWTQVGVSQGHNMCPDPGIPERGKRLGSDFRL
SVQFTCNEGYDLQGSKRITCMKVSDMFAAWSDHRPVCRARMCDAHLRGPSGIITSPNFPIQYDNNA
ITALNPSKVIKLAFEEFDLERGYDTLTVGDGGQDGDQKTVLYMLTGTSVPDLIVSTNHQMWLL
F
GSSLGFKASYEEIEQGSCGDPGIPAYGRREGSRFRHGDTLKFECQPAFELVGQKAITCQKNN
PSGWLSPNYPEDYGNHLHCVWLILARPESRIHLAFNDIDVEPQFDFLV
KDGATAEAPVLGTFSGNQLPSSITSSGHVARLEFQTDHSTGKRGFNITFTSESSNECPDPGVPVNGK
ETITCVLKEGSVVWNSAVLRCEAPCGGHLTSPSGTILSPGWP
G
FYKDALSCAWVIEAQPGYPIKITFDRFKTEVNYDTLEVRDGRTYSAPLIGVYHGTQVPQFLISTSNY
STDKSHSDIGFQLRYEAITLQSDHCLDPGIPVNGQRHGNDFWGALVTFSCDSGYTLSDGEPL
SCEALCGGFIQGSSGTILSPGFPDFYPNNLNCTWIIETSHGKGVFFTFHTFHLES
ITENGSFTQPLRQLTGSRLPAPISAGLYGNFTAQVRFISDFSMSYEGFNITFSEYDLEPCEE
AYSIRKGLQFGVGDTLTFSCFPGYRLEGTARITCLGGRRRLWSSPLPRCVAECGNSVTGTQGTL
IQTQPGKGIQLKARAFELSEGDVLKVYDGNNNSARLLGVFSHSEMMGVTL
SSLWLDFITDAENTSKGFELHFSSFELIKCEDPGTPKFGYKVHDEGHFAGSSVSFSCDPGYSLR
TWDRPLPTCVAECGGTVRGEVSGQVLSPGYPAPYEHNLNCIWTIEAEAGCTIGLH
GVLLKELSGPALPKDLHSTFNSWLQFSTDFFTSKQGFAIQFSGST
CNDPGIPQNGSRSGDSWEAGDSTVFQCDPGYALQGSAEISCVKIENRFFWQPSPPTCIAPCGGDL
PGKECDWKVTVSPDWIALVLFPSFNLEPGYDFLHIYDGRDSLSPLIGSFY
SQLPGRIESSSNSLFLAFRSDASVSNAGFVIDFPENPRESCFDPGSIKNGTRVGSDLKLGSSVTWC
STLSCILGPDGKPVWNNPRPVCTAPCGGQWGSDGWLSPNYPQNYTSGQICLYFVTVP
K
QHSRLLSSLSGSHTGESLPLATSNQVLIKFSAKGLAPARG
F
VPRTSATQCSSVPEPRYGKRLGSDFSVGAIVRFECNSGYALQGSPEIECLPVPGALAQWNV
IQIQWSFVTEQNWDSLEVFDG
159



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
FSGTTVPALLNSTSNQLYLHFYSDISVSAAGFHLEYKTVGLSSCPEPAVPSNGVKTGE
FQCEPGYALQGHAHISCMPGTVRRWNYPPPLCIAQCGGTVEEMEGVILSPGFPGNYPSN
STEPNHDYIEIRNGPYETSRMMGRFSGSELPSSLLSTSHETTVYFH
ITSSNGTVYSPGFPSPYSSSQDCVWLITVPIGHGVRLNLSLLQTEPSGDFIT
SSNQVLLKFHRDAATGGIFAIAFSAYPLTKCPPPTILPNAEV
CEVHCPTNELLTDSTGVILSQSY
SLTVEYFLSEKQYDEFEIFDGPSGQSPLLKALSGNYSAPLIVTSSS
SVYLRWSSDHAYNRKGFKIRYSAPYCSLPRAPLHGFILGQTSTQPGGSIHFGCNAGYRLVGHSMAIC
T
CSEGYHLQAGAEATAECLDTG
SISVEHGRWRLIFETQYQFQAQLMLICDPGYYYTGQRVIRCQANGKWS
IPPNGHRIGTLSVYGATATFSCNSGYTLVGSRVRECMANGLWSGSEVRCLA
IVNGHINGENYSYRGSVVYQCNAGFRLIGMSVRICQQDHHWSGKTPFCVPITCGHPGNPV
INCTDPGHQENSVRQVHASG
FYLLGTPVLSCQGDGTWDRPRPQCLLVSCGHPGSPPHSQMSGDSYTVGAVV
CIGKRTLVGNSTRMCGLDGHWTGSLPHCSGTSVGVCGDPGIPAHGTRLGDSFDPGTVMRFSCEAG
SERTCQANGSWSGSQPECGVISCGNPGTPSNARVVFSDGLVFSSSIVYECREGYYATGLLSR
CSVNGTWTGSDPECLVINCGDPGIPANGLRLGNDFRYNKTVTYQCVPGYMMESHRVSVLSCTKDRTW
IPNGKWGSDFMWGSSVTYACLEGYQLSLPAVFTCEGNGSWTGELPQCFPV
SCHPPLVLVGSPRRFCQSDGTWSGTQPSCIDPTLTTCADPGV
STVLFRCQKGYLLQGSTTRTCLPNLTWSGTPPDCVPHHCRQPETPTHANVGAL
IYSCQEGFSLKGGSEHRTCKADGSWTGKPPICLEVRPNGRPINTAREPPLTQALIPGDV
KKQPAMLRVTGFQVANSKVNATMIDHSGVELHLAGTYKKEDFHLLLQVYQITG
VEIFMNKFKDDHWALDGHVSSESSGATFIYQGSVKGQGFGQFGFQRLDLRLLESDPESIGRHFASNS
S
SVAAAILVPFIALIIAGFVLYLYKHRRRPKVPFNGYAGHENTNVRATFENPMYDRNIQPTDIMASEA
FTVSTVCTAV
lOV4b, CG50377-Ol SEQ ID NO: 3310136 bp_ _
>NA Sequence ORF Start: ATG at 1 ORF Stop: TGA at 9313
TGGCGGGCGCCCCTCCCCCCGCCTTGCTGCTGCCTTGCAGTTTGATCTCAGACTGCTGTGCTAGCA
160



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TAATATGTGTCCAGACCCTGGCATACCCGAAAGGGGCAAAAGACTAGGCTCGGATTTCA
TCCAGCGTCCAGTTCACCTGCAACGAGGGCTATGACCTGCAAGGGTCCAAGCGGATCACC
TGTTTGCGGCCTGGAGCGACCACAGGCCAGTCTGCCGAGCCCGCATGTG
CCACCTTCGAGGCCCCTCGGGCATCATCACCTCCCCCAATTTCCCCATTCAGTATGACAACA
TCAAGCTCGCCTTTGAGGAG
TGGTGGTCAGGATGGGGACCAGAAGAC
ACATGTCTCAAAATGCCTGCAGTGACAGCCCTCACACCCCAGGCTCTCGCATCCCAGAGA
TCTGGAGGCAGAAATGGACTGTACTTGAGATCTGTCGTGACATTAGCAGTTCA
TGCCAAAAGAATAACCAATGGTCGGCTAAGAAGCCAGGCTGCGTGTTCTCCTGCTTCTTCAA
TGTCTGGCTCATCCTGGCCAGGCCTGAGAGCCGCATCCACCTGGCCTTCAACGACATTGACGTGGAG
C
CTCAGTTTGATTTCCTGGTCATCAAGGATGGGGCCACCGCCGAGGCGCCCGTCCTGGGCACCTTCTC
TCACAAGCAGTGGCCACGTGGCCCGTCTCGAGTTCCAGACTGACC
AAATGGCAAACGGTTTGGGGACAGCCTCCAGCTGGGCAGCTCCATCTCCTTCCTCTG
TGAAGGCTTCCTTGGGACTCAGGGCTCAGAGACCATCACCTGCGTCCTGAAGGAGGGCAGCGTGG
TCCTCTCTCCGGGCTGGCCTGGCTTCTACAAGGATGCCTTGAGCTGTGCCTGGGTGATTGAGGCCCA
G
CCAGGCTACCCCATCAAAATCACCTTCGACAGATTCAAAACCGAGGTCAACTATGACACCCTGGAAG
ACTCAGCGCCCTTGATCGGGGTTTACCACGGGACCCAGGTTCCCCAGTTC
ACCTCTACCTCCTCTTCTCTACCGACAAGAGTCACTCGGACATCGGCTT
AACACTGCAGTCAGACCACTGTCTGGATCCAGGAATCCCAGTAAATG
TTCAAGGCTCCAGTGGGACCATCTTGTCGCCAGGGTTCCCTGACTTCT
TGGCAAGGGTGTGTTCTTCACTTTC
161



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TCTCGGCTGCCAGCTCCCATCAGCGCTGGGCTCTATGGCAACTTCACTG
TCTCTGATTTCTCCATGTCATATGAAGGATTCAACATCACCTTCTCAGAGTAC
TGCAT
CCGTTTGCTGGGAGTTTTTAGCCATTCT
.TGGGGGTGACTTTGAACAGCACATCCAGCAGTCTGTGGCTTGATTTCATCACTGATGCTGA
TGAAGGTCATTTTGCAGGGAGCTCCGTGTCCTTCAGCTGT
CGGCCTCTGCCCACCTGTGTCGCCGAGTGTGGAGGGACAGTGAGAGGAGAGGTGTCGGGGCAGGTGC
GATGGGCCTGTGGAGAGCGGGGTTCTGCTGAAGGAGCTGAGTGGCCCGGCCCTGCCCAAGGACCTGC
AGCACCTTCAACTCGGTCGTCCTGCAGTTCAGCACTGACTTCTTCACCAGCAAGCAGGGCTTTGCC
TCCCGCAGAATGGGAGTCGGAG
TC
CCGCCAGGCAAGGAGTGTGACTGGAAAGTGACCGTCTCACCAGACTACGTCATCGCCCTGGTATTTA
TATCTACGACGGACGGGACTCTCTCAGCCCT
TCTGTGAGCAATGCTGGCTTCGTCATTGACTATACAGAAAACCCGCGGG
TGTTTTGATCCTGGTTCCATCAAGAACGGCACACGGGTGGGGTCCGACCTGAAGCTGGGCTCC
ACTACTGCCACGGGGGCTACGAAGTTGAGGGCACCTCGACCCTGAGCTGCATCCTGGG
TGGGAAGCCCGTGTGGAACAATCCCCGGCCAGTCTGCACAGCCCCCTGTGGGGGACAGTATG
CAACTACCCCCAGAACTACACCAGTGGACAGATCTGCTTG
TGTGGTGTTTGGCCAGTTCGCCTTCTTTCACACGGCCCTCAA
162



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CC
GTGCCGGAACCCCGCTATGGCAAGAGGCTGGGCAGTGACTTCTCGGTGGGGGCCATCGTCCGCTTCG
A
ATGCAACTCCGGCTATGCCCTGCAGGGGTCGCCAGAGATCGAGTGCCTCCCTGTGCCTGGGGCCTTG
G
CCCAATGGAATGTCTCAGCGCCCACGTGTGTGGTGCCGTGTGGAGGCAACCTCACAGAGCGCAGGGG
C
ACCATCCTGTCCCCTGGCTTCCCAGAGCCGTACCTCAACAGCCTCAACTGTGTGTGGAAGATCGTGG
AACCATGCTGGGGAGTTTCTCAGGAACAACCGTGCCTGC
TGAAGACTGGCGAGCGCTACTTGGTGAATGATGTGGTGTCTTTCCAGTGTGAGCCGGGATATGCCCT
C
CAGGGCCACGCCCACATCTCCTGCATGCCCGGAACAGTGCGGCGATGGAACTACCCTCCTCCACTCT
G
TATTGCACAGTGTGGGGGAACAGTGGAGGAGATGGAGGGGGTGATCCTGAGCCCCGGCTTCCCAGGC
TCCTGAACTTCTCCACCGAGCCCAACCACGACTACATAGAAATCCGGAATGGCCCCTATGAGACCA
CGCATGATGGGAAGATTCAGTGGAAGCGAGCTTCCAAGCTCCCTCCTCTCCACGTCCCACGAGACC
CGTGTATTTCCACAGCGACCACTCCCAGAATCGGCCAGGATTCAAGCTGGAGTATCAGGCCTATGA
TTCAAGAGTGCCCAGACCCAGAGCCCTTTGCCAATGGCATTGTGAGGGGAGCTGGCTACAACGTGG
TGGCACCAACCGGAACTGGGACCACCCCCTGCCCAAGTGTGAAGTCCCTTGTGGCGGGAACATCAC
GAGATTTCATCACCATCTGGGATGGGCCACAGCAAACAGCACCACGGCTCGGCGTCTTCACCCGGAG
C
ATGGCCAAGAAAACAGTGCAGAGTTCATCCAACCAGGTCCTGCTCAAGTTCCACCGTGATGCAGCCA
C
AGGGGGGATCTTCGCCATAGCTTTCTCCGCTTATCCACTCACCAAATGCCCTCCTCCCACCATCCTC
CTTTACCTTAGTGGGGAATGAAATTCTGACCTGCAAACTTGGAACCTACCTGCAGTTTGAAG
CCCCGATATGTGAAGTGCACTGTCCAACAAATGAGCTTCTGACAGACTCCACAGGCGTGATC
CAGAGCTACCCTGGAAGCTATCCCCAGTTCCAGACCTGCTCTTGGCTGGTGAGAGTGGAGCC
.TAACATCTCCCTCACAGTGGAGTACTTCCTCAGCGAGAAGCAATATGATGAGTTTGAGATTT
163



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TGATGGTCCATCAGGACAGAGTCCTCTGCTGAAAGCCCTCAGTGGGAATTACTCAGCTCCCCTGATT
TCACGCCTACAATCGGAAGGGCTT
C
TGGAATGGTGTTTGGCAAGGAGTACACAGTGG
TGTCCCACCACAGTGTGTCCCTGTGACTTGTCCTGA
TCAGTTCCAGG
TGGAGCCTCGGGGACTCTACGCCCACCTGCCGAATCATCTCCTGTGGAGAGCTCCCGAT
TGGCCACCGCATCGGAACACTGTCTGTCTACGGGGCAACAGCCATCTTCTCCTGCAATT
ACACACTGGTGGGCTCCAGGGTGCGTGAGTGCATGGCCAATGGGCTCTGGAGTGGCTCTGAA
TCCGCTGCCTTGCTGGACACTGTGGGACTCCTGAGCCCATTGTCAACGGACACATCAATGGGGAGAA
C
TACAGCTACCGGGGCAGTGTGGTGTACCAATGCAATGCTGGCTTCCGCCTGATCGGCATGTCTGTGC
G
CATCTGCCAGCAGGATCATCACTGGTCGGGCAAGACCCCTTTCTGTGTGCCAATTACCTGTGGACAC
C
CAGGCAACCCTGTCAACGGCCTCACTCAGGGTAACCAGTTTAACCTCAACGATGTGGTCAAGTTTGT
TATGGCTGAGGGGGCTGCTAGGTCCCAATGCCTGGCCAGCGGGCAATGGAGTG
TGCTGCCCACCTGCAGAATCATCAACTGTACAGATCCTGGACACCAAGAAAATAGTGTTCGTCAG
CCGCACAGGTTCAGCTTCGGCACCACTGTGTCTTACCGGTGCAACCACGGCTT
TCGGCAAGCGTACTCTGGTGGGAAACAGCACCCGCATGTG
CCCTGGGATCCCGGCTCATGGCATCCGTTTGGGGGACAGCTTTGATCCAGGCACTGTGATGCGCTTC
TGGCTCGTGGAG
C
TGCCCGAGTTG
CTGGTTTTCTCCAGCTCTATCGTCTATGAGTGCCGGGAAGGATACTACGCCACA
164



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
C
TGTGCAAGCCACCTCCGCTCAT
C
-- - ---""""""'-n ~ ~~H~ 1 ~~~H~~c'AGCCCAGCTGCATAGATCCGACCCTGACCACGTGT
G
CGGACCCTGGTGTGCCACAGTTTGGGATACAGAACAATTCTCAGGGCTACCAGGTTGGAAGCACAGT
C
CTCTTCCGTTGTCAAAAAGGCTACCTGCTTCAGGGCTCCACCACCAGGACCTGCCTCCCAAACCTGA
C
CTGGAGTGGAACCCCACCTGACTGTGTCCCCCACCACTGCAGGCAGCCAGAGACGCCAACGCATGCC
C
TGAATACCAGGGGAAGAAGCAG
~.~.-~-1 ~ i v~ta~;wry:'1°1"1'ATGAATAAGTTCAAAGATGATCACTGGGCTTTAGATGGC
C
CAGTTCGGCTTTCAAAGACTGGACCTCAGGCTGCTGGAGTCAGACCCCGAGTCCATTGGCCGCCACT
T_
rGCTTCCAACAGCAGCTCAGTGGCAGCCGCGATCCTGGTGCCTTTCATCGCCCTCATTATTGCGGGC
r
TGTACGACCGCAACATCCAGCCCACAGACATCA
-r""-H~.~~ 1 ~ 1 c,c-rac.AGCAGTATAGCCACCCGGCCTGGCCGCT
T_
TTTTTGCTAGGTTGAACTGGTACTCCAGCAGCCGCCGAAGCTGGACTGTACTGCTGCCATCTCAGCT
C
-.~::~s.r:.~.
4b, CG50377-Ol SEQ m NO: 34'3104 as MW at 336746.3kD
.r c~........___
LLPCSLISDCCASNQRHSVGVGPSELVKKQIELKSRGVKLMPSKDNSQKTSVLTQVGV
GIPERGKRLGSDFRLGSSVQFTCNEGYDLQGSKRITCMKVSDMFAAWSDHRPVCRARM
IITSPNFPIQYDNNAHCVWIITALNPSKVIKLAFEEFDLERGYDTLTVGDGGQDGDQK
165



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
QNACSDSPHTPGSRIPESMSGDIWRQKWTVLEICRDISSSDARSGSVRKSPKTSNAVELVAP
IPAYGRREGSRFHHGDTLKFECQPAFELVGQKAITCQKNNQWSAKKPGCVFSCFF
PSGWLSPNYPEDYGNHLHCVWLILARPESRIHLAFNDIDVEPQFDFLVIKDGATAEAPVLGTF
SGHVARLEFQTDHSTGKRGFNITFTTFRHNECPDPGVPVNGKRFGDSLQLGSSISFL
VLRCEAPCGGHLTSPSGTILSPGWPGFYKDALSCAWVIEA
PGYPIKITFDRFKTEVNYDTLEVRDGRTYSAPLIGVYHGTQVPQFLISTSNYLYLLFSTDKSHSDIG
QLRYETITLQSDHCLDPGIPWGQRHGNDFWGALVTFSCDSGYTLSDGEPLECEPNFQWSRALPSC
FIQGSSGTILSPGFPDFYPNNLNCTWIIETSHGKGVFFTFHTFHLESGHDYLLITENGSFTQ
SAGLYGNFTAQVRFISDFSMSYEGFNITFSEYDLEPCEEPEVPAYSIRKGLQFG
CFPGYRLEGTARITCLGGRRRLWSSPLPRCVAECGNSVTGTQGTLLSPNFPVNYNNNHEC
IQTQPGKGIQLKARAFELSEGDVLKVYDGNNNSARLLGVFSHSEMMGWLNSTSSSLWLDFITDA
KGFELHFSSFELIKCEDPGTPKFGYKVHDEGHFAGSSVSFSCDPGYSLRGSEELLCLSGERRTW
EVSGQVLSPGYPAPYEHNLNCIWTIEAEAGCTIGLHFLVFDTEEVHDVLRI
PALPKDLHSTFNSVVLQFSTDFFTSKQGFAIQFSVSTATSCNDPGIPQNGSR
ISCVKIENRFFWQPSPPTCIAPCGGDLTGPSGVILSPNYPEP
PPGKECDWKVWSPDWIALVFNIFNLEPGYDFLHIYDGRDSLSPLIGSFYGSQLPGRIESSSNSLF
IKNGTRVGSDLKLGSSVTYYCHGGYEVEGTSTLSCIL
PDGKPVWNNPRPVCTAPCGGQYVGSDGVVLSPNYPQNYTSGQICLYFVTVPKDYWFGQFAFFHTAL
QHSRLLSSLSGSHTGESLPLATSNQVLIKFSAKGLAPARGFHFWQAVPRTSATQCS
FSVGAIVRFECNSGYALQGSPEIECLPVPGALAQWNVSAPTCWPCGGNLTERR
ILSPGFPEPYLNSLNCVWKIWPEGAGIQIQWSFVTEQNWDSLEVFDGADNTVTMLGSFSGTTVP
YLHFYSDISVSAAGFHLEYKTVGLSSCPEPAVPSNGVKTGERYLVNDWSFQCEPGYA
SCMPGTVRRWNYPPPLCIAQCGGWEEMEGVILSPGFPGNYPSNMDCSWKIALPVGFGAHI
TEPNHDYIEIRNGPYETSRMMGRFSGSELPSSLLSTSHETTVYFHSDHSQNRPGFKLEYQAY
SNGTWSPGFPSPYSSSQDCVWLITVPIGHGVRLNLSLLQTEPSGDFITIWDGPQQTAPRLGVFTR
SAYPLTKCPPPTILPNAEVWENEEFNIGDIVRYRC
~VGNEILTCKLGTYLQFEGPPPICEVHCPTNELLTDSTGVILSQSYPGSYPQFQTCSWLVRVE
IFDGPSGQSPLLKALSGNYSAPLIVTSSSNSWLRWSSDHAYNRKG
SAPYCSLPRAPLHGFILGQTSTQPGGSIHFGCNAGYRLVGHSMAICTRHPQGYHLWSEAIPLC
166



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CGLPEAPKNGMVFGKEYTVGTKAWSCSEGYHLQAGAEATAECLDTGLWSNRNVPPQCVPVTCP
VSSTSVEHGRWRLIFETQYQFQAQLMLICDPGYWTGQRVIRCQANGKWSLGDSTPTCRIISCGELP
I
PPNGHRIGTLSWGATAIFSCNSGYTLVGSRVRECMANGLWSGSEVRCLAGHCGTPEPIVNGHINGE
GKTPFCVPITCGHPGNPVNGLTQGNQFNLNDWKF
QCLASGQWSDMLPTCRIINCTDPGHQENSVRQVHASGPHRFSFGTTVSYRCNHG
PPHSQMSGDSYTVGAVVRYSCIGKRTLVGNSTRM
LPHCSGTSVGVCGDPGIPAHGIRLGDSFDPGTVMRFSCEAGHVLRGSSERTCQANGSW
CGVISCGNPGTPSNARWFSDGLVFSSSIWECREGYYATGLLSRHCSVNGTWTGSDPECLV
IPANGLRLGNDFRYNKTVTYQCVPGYMMESHRVSVLSCTKDRTWNGTKPVCKALMCKPPPL
PNGKWGSDFMWGSSVTYACLEGYQLSLPAVFTCEGNGSWTGELPQCFPVFCGDPGVPSRGRREDRG
F
SYRSSVSFSCHPPLVLVGSPRRFCQSDGTWSGTQPSCIDPTLTTCADPGVPQFGIQNNSQGYQVGST
LFRCQKGYLLQGSTTRTCLPNLTWSGTPPDCVPHHCRQPETPTHANVGALDLPSMGYTLITPARRAS
P
SRVAPSTAPARRMAAGQASRPSAWRSGPVGDPSTLPGSHRSPKP
V4c, CG50377-02 SEQ m N0: _35 8010 by
A Sequence _~_'_O~ Start: ATG at 1 ORF Stop: TAG at 8008
AGCA
TGCCCAGCAAAGACAACAGCCAGAAGACGTCTGTGTTAACTCAGGTTGGTGTGTC
TTTCA
CTGCAACGAGGGCTATGACCTGCAAGGGTCCAAGCGGATCACC
ATGAAAGTGAGCGACATGTTTGCGGCCTGGAGCGACCACAGGCCAGTCTGCCGAGCCCGCATGTG
TCATCACCTCCCCCAATTTCCCCATTCAGTATGACAACA
TCATCACAGCACTCAACCCCTCCAAGGTGATCAAGCTCGCCTTTGAGGAG
TGACACCCTGACGGTCGGTGATGGTGGTCAGGATGGGGACCAGAAGAC
TGCCTGCAGTGACAGCCCTCACACCCCAGGCTCTCGCATCCCAGAGA
TAACCAATGGTCGGCTAAGAAGCCAGGCTGCGTGTTCTCCTGCTTCTTCAA
ACCCAGAGGACTATGGCAACCACCTCCACT
167



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CATCACAAGCAGTGGCCACGTGGCCCGTCTCGAGTTCCAGACTGACC
G
TGGCAAACGGTTTGGGGACAGCCTCCAGCTGGGCAGCTCCATCTCCTTCCTCTG
TTGGGACTCAGGGCTCAGAGACCATCACCTGCGTCCTGAAGGAGGGCAGCGTGG
TCCTCTCTCCGGGCTGGCCTGGCTTCTACAAGGATGCCTTGAGCTGTGCCTGGGTGATTGAGGCCCA
G
CCAGGCTACCCCATCAAAATCACCTTCGACAGATTCAAAACCGAGGTCAACTATGACACCCTGGAAG
T
ACGCGATGGGCGGACTTACTCAGCGCCCTTGATCGGGGTTTACCACGGGACCCAGGTTCCCCAGTTC
ACTATAACACTGCAGTCAGACCACTGTCTGGATCCAGGAATCCCAGTAAATG
TGACTTCTACGTGGGCGCGCTGGTGACCTTCAGCTGTGACTCGGGCTACACA
GCTCTCTGTGGTGGCTTCATTCAAGGCTCCAGTGGGACCATCTTGTCGCCAGGGTTCCCTGACTTCT
A
CCCCAACAACTTGAACTGCACCTGGATTATCGAAACATCTCATGGCAAGGGTGTGTTCTTCACTTTC
TCTCGGCTGCCAGCTCCCATCAGCGCTGGGCTCTATGGCAACTTCACTG
TGATTTCTCCATGTCATATGAAGGATTCAACATCACCTTCTCAGAGTAC
T
GGGGGGCAGACGGCGCCTGTGGAGCTCGCCTCTGCCAAGGTGTGTTGCTGAGTGTGGGAATTCAGTC
A
CAGGCACTCAGGGTACTTTGCTGTCCCCCAACTTTCCTGTGAACTACAATAACAATCATGAATGCAT
TTCT
AACACCAGCAAGGGCTTTGAACTGCACTTTTCCAGCTTTGAACTCATCAAATGTGAGGACCCAGGAA
C
CCCCAAGTTTGGCTACAAGGTTCATGATGAAGGTCATTTTGCAGGGAGCTCCGTGTCCTTCAGCTGT
G
ACCCTGGATACAGCCTGCGGGGTAGTGAGGAGCTGCTGTGTCTGAGTGGAGAGCGCCGGACCTGGGA
TCCAGCTCCCTATGAACACAATCTCAACTGCATCTGGACCATCGAAGCAGAGGCC
168



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TTGGGCTACACTTCCTGGTGTTTGACACAGAGGAGGTTCACGACGTGCTGCGCATCTG
TGACCCTGGGATCCCGCAGAATGGGAGTCGGAG
TTACCCAGAACCCTA
CCGCCAGGCAAGGAGTGTGACTGGAAAGTGACCGTCTCACCAGACTACGTCATCGCCCTGGTATTTA
T
CCTGGCTATGACTTCCTCCATATCTACGACGGACGGGACTCTCTCAGCCCT
TCATAGGAAGCTTCTATGGCTCCCAGCTCCCAGGCCGCATTGAAAGCAGCAGCAACAGCCTCTTCCT
C
TGCTGGCTTCGTCATTGACTATACAGAAAACCCGCGGG
TCAAGAACGGCACACGGGTGGGGTCCGACCTGAAGCTGGGCTCC
CTGAGCTGCATCCTGGG
CCTGATGGGAAGCCCGTGTGGAACAATCCCCGGCCAGTCTGCACAGCCCCCTGTGGGGGACAGTATG
ACCCCCAGAACTACACCAGTGGACAGATCTGCTTG
ATTTTGTTACTGTGCCCAAGGACTATGTGGTGTTTGGCCAGTTCGCCTTCTTTCACACGGCCCTCAA
C
TCACTGCCCTTGGCCACCTCCAATCAAGTTCTCATTAAGTTCAGCGCCAAAGGCCTC
CACCAGCCAGAGGCTTCCACTTTGTCTACCAAGCGGTTCCTCGAACCAGCGCCACGCAGTGCAGCTC
T
TGCAACTCCGGCTATGCCCTGCAGGGGTCGCCAGAGATCGAGTGCCTCCCTGTGCCTGGGGCCTTG
CCCAATGGAATGTCTCAGCGCCCACGTGTGTGGTGCCGTGTGGAGGCAACCTCACAGAGCGCAGGGG
C
TCCAGATCCAAGTTGTCAGTTTTGTGACAGAGCAGAACTGGGACTCGCTG
TTTGATGGTGCAGATAACACTGTAACCATGCTGGGGAGTTTCTCAGGAACAACCGTGCCTGC
TTTCTACTCAGATATCAGCGTATCTGCAGCTG
TGCCCGGAACAGTGCGGCGATGGAACTACCCTCCTCCACTCT
TATTGCACAGTGTGGGGGAACAGTGGAGGAGATGGAGGGGGTGATCCTGAGCCCCGGCTTCCCAGGC
169



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CGAGCCCAACCACGACTACATAGAAATCCGGAATGGCCCCTATGAGACCA
TCGGCCAGGATTCAAGCTGGAGTATCAGGCCTATGA
CTTCAAGAGTGCCCAGACCCAGAGCCCTTTGCCAATGGCATTGTGAGGGGAGCTGGCTACAACGTGG
ACAATCAGTGACCTTCGAGTGCCTCCCGGGGTATCAATTGACTGGCCACCCTGTCCTCACGTGTCAA
C
ATGGCACCAACCGGAACTGGGACCACCCCCTGCCCAAGTGTGAAGTCCCTTGTGGCGGGAACATCAC
CCTAGCCCGTACTCCAGCTCCCAGGACTGTGTCT
C
CAAGAAAACAGTGCAGAGTTCATCCAACCAGGTCCTGCTCAAGTTCCACCGTGATGCAGCCA
TAGCTTTCTCCGCTTATCCACTCACCAAATGCCCTCCTCCCACCATCCTC
C
CCAACGCCGAAGTCGTCACAGAGAATGAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCT
C
CCTGGCTTTACCTTAGTGGGGAATGAAATTCTGACCTGCAAACTTGGAACCTACCTGCAGTTTGAAG
CTCACAGTGGAGTACTTCCTCAGCGAGAAGCAATATGATGAGTTTGAGATTT
TT
CTACAATCGGAAGGGCTT
TTCAGCCCCTTACTGCAGCCTGCCCAGGGCTCCACTCCATGGCTTCATCCTAGGCC
TCCACTTTGGCTGCAACGCCGGCTACCGCCTGGTGGGACAC
TGGCCATCTGTACCCGGCACCCCCAGGGCTACCACCTGTGGAGCGAAGCCATCCCTCTCTGTCA
G
TGTCCCACCACAGTGTGTCCGTGAGTCCTCGGGCAA
.TCAGCGTGGAGCATGGCCGATGGAGGCTTA
ATCAGTTCCAGGCCCAGCTGATGCTCATCTGTGACCCTGGCTACTACTATACT
170



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CCAATGGGCTCTGGAGTGGCTCTGAAGTCCGCTGCCTTGCCACTCAGACCAAGCTCCACTCCATTTT
TCAATGGGGAGAACTACAGCTACCGGGGCAGTGTGGTGTACCAATGC
TGTTAAGCAGCAGTTGCTGCTGCTGCTGCTGCTGTTGTGTGATGATGATG
TGATGGTAGTGGTGCAATTACCTGTGGACACCCAGGCAACCCTGTCAACGGCCTCACT
AGGTCCCAATGCCTGGCCAGCGGGCAATGGAGTGACATGCTGCCCACCTGCAGAATCATCA
TCCTGGACACCAAGAAAATAGTGTTCGTCAGGTCCACGCCAGCGGCCCGCACAGGTTC
C
TGCCAGGGAGATGGCACATGGGACCGTCCCCGCCCCCAGTGTCTCTGTAAGTAG
4c, CG50377-02 ~SEQ ID NO: 36 2669 as MW at 290805.9kD
in Seauence
SELVKKQIELKSRGVKLMPSKDNSQKTSVLTQVGV
S
QGHNMCPDPGIPERGKRLGSDFRLGSSVQFTCNEGYDLQGSKRITCMKVSDMFAAWSDHRPVCRARM
C
DAHLRGPSGIITSPNFPIQYDNNAHCVWIITALNPSKVIKLAFEEFDLERGYDTLTVGDGGQDGDQK
T
VLYMSQNACSDSPHTPGSRIPESMSGDIWRQKWTVLEICRDISSSDARSGSVRKSPKTSNAVELVAP
G
TEIEQGSCGDPGIPAYGRREGSRFHHGDTLKFECQPAFELVGQKAITCQKNNQWSAKKPGCVFSCFF
N
FTSPSGVVLSPNYPEDYGNHLHCVWLILARPESRIHLAFNDIDVEPQFDFLVIKDGATAEAPVLGTF
PSSITSSGHVARLEFQTDHSTGKRGFNITFTTFRHNECPDPGVPVNGKRFGDSLQLGSSISFL
ETITCVLKEGSVVWNSAVLRCEAPCGGHLTSPSGTILSPGWPGFYKDALSCAWVIEA
TYSAPLIGVYHGTQVPQFLISTSNYLYLLFSTDKSHSDIG
QLRYETITLQSDHCLDPGIPVNGQRHGNDFYVGALVTFSCDSGYTLSDGEPLECEPNFQWSRALPSC
E
ALCGGFIQGSSGTILSPGFPDFYPNNLNCTWIIETSHGKGVFFTFHTFHLESGHDYLLITENGSFTQ
SAGLYGNFTAQVRFISDFSMSYEGFNITFSEYDLEPCEEPEVPAYSIRKGLQFG
GDTLTFSCFPGYRLEGTARITCLGGRRRLWSSPLPRCVAECGNSVTGTQGTLLSPNFPVNYNNNHEC
I
YSIQTQPGKGIQLKARAFELSEGDVLKVYDGNNNSARLLGVFSHSEMMGVTLNSTSSSLWLDFITDA
SSFELIKCEDPGTPKFGYKVHDEGHFAGSSVSFSCDPGYSLRGSEELLCLSGERRTW
SPGYPAPYEHNLNCIWTIEAEAGCTIGLHFLVFDTEEVHDVLRI
STDFFTSKQGFAIQFSVSTATSCNDPGIPQNGSR
SGVILSPNYPEP
171



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
PLIGSFYGSQLPGRIESSSNSLF
SCFDPGSIKNGTRVGSDLKLGSSVTYYCHGGYEVEGTSTLSCIL
ICLYFVTVPKDYWFGQFAFFHTAL
SLSGSHTGESLPLATSNQVLIKFSAKGLAPARGFHFWQAVPRTSATQCS
IECLPVPGALAQWNVSAPTCWPCGGNLTERR
LSPGFPEPYLNSLNCVWKIWPEGAGIQIQWSFVTEQNWDSLEVFDGADNTVTMLGSFSGTTVP
TSNQLYLHFYSDISVSAAGFHLEYKWGLSSCPEPAVPSNGVKTGERYLVNDWSFQCEPGYA
QGHAHISCMPGTVRRWNYPPPLCIAQCGGTVEEMEGVILSPGFPGNYPSNMDCSWKIALPVGFGAHI
n
IRNGPYETSRMMGRFSGSELPSSLLSTSHETTWFHSDHSQNRPGFKLEYQAY
CPDPEPFANGIVRGAGYNVGQSVTFECLPGYQLTGHPVLTCQHGTNRNWDHPLPKCEVPCGGNI
SSNGTWSPGFPSPYSSSQDCVWLIWPIGHGVRLNLSLLQTEPSGDFITIWDGPQQTAPRLGVFTR
a
FNIGDIVRYRC
PICEVHCPTNELLTDSTGVILSQSYPGSYPQFQTCSWLVRVE
SLTVEYFLSEKQYDEFEIFDGPSGQSPLLKALSGNYSAPLIVTSSSNSWLRWSSDHAYNRKG
nirc x 5t-~r x t.;51~Y1tAPLHGFILGQTSTQPGGS IHFGCNAGYRLVGHSMAI CTRHPQGYHLWSEAI
PLC
Q
SISVEHGRWRLIFETQYQFQAQLMLICDPGWYTGQRVIRCQANGKWSLGDSTPTC
ISCGELPIPPNGHRIGTLSWGATAIFSCNSGYTLVGSRVRECMANGLWSGSEVRCLATQTKLHSI
KLLFDVLSSPSLTKAGHCGTPEPIVNGHINGENYSYRGSWYQCNAGFRLIGMSVRICQQDHHWSG
G
GQWSDMLPTCRIINCTDPGHQENSVRQVHASGPHRFSFGTTVSYRCNHGFYLLGTPVL
V4d, CG50377-03 SEQ m NO: 37 995,1_b_p _
A Sequence ORF Start: ATG at 1 ORF Stop: TGA at 7837
TCTCAGACTGCTGTGCTAGCA
TCAGCGACACTCCGTGGGCGTAGGACCCTCCGAGCTAGTCAAGAAGCAAATTGAGTTGAAGTCTCGA
n
AATATGTGTCCAGACCCTGGCATACCCGAAAGGGGCAAAAGACTAGGCTCGGATTTCA
TGACCTGCAAGGGTCCAAGCGGATCACC
TGTG
TCACCTCCCCCAATTTCCCCATTCAGTATGACAACA
172



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TCAAGCTCGCCTTTGAGGAG
TTTGGAGAGGGGCTATGACACCCTGACGGTCGGTGATGGTGGTCAGGATGGGGACCAGAAGAC
CATGTCTGGGGACATCTGGAGGCAGAAATGGACTGTACTTGAGATCTGTCGTGACATTAGCAGTTCA
TTCACCAGCCCGTCTGGGGTTGTCCTGTCTCCCAACTACCCAGAGGACTATGGCAACCACCTCCACT
TCCACCTGGCCTTCAACGACATTGACGTGGAG
TGGGGCCACCGCCGAGGCGCCCGTCCTGGGCACCTTCTC
CAGACTGACC
GCGTTCCAGTAAATGGCAAACGGTTTGGGGACAGCCTCCAGCTGGGCAGCTCCATCTCCTTCCTCTG
T
GATGAAGGCTTCCTTGGGACTCAGGGCTCAGAGACCATCACCTGCGTCCTGAAGGAGGGCAGCGTGG
T
CAGTTC
TCAGCACCAGCAACTACCTCTACCTCCTCTTCTCTACCGACAAGAGTCACTCGGACATCGGCTT
AACACTGCAGTCAGACCACTGTCTGGATCCAGGAATCCCAGTAAATG
AAGTGACGGGGAGCCTCTGGAGTGTGAGCCCAACTTCCAGTGGAGCCGGGCCCTGCCCAGTTGTGA
TTATCGAAACATCTCATGGCAAGGGTGTGTTCTTCACTTTC
TCTCGGCTGCCAGCTCCCATCAGCGCTGGGCTCTATGGCAACTTCACTG
TCACCTTCTCAGAGTAC
173



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CAGGCACTCAGGGTACTTTGCTGTCCCCCAACTTTCCTGTGAACTACAATAACAATCATGAATGCAT
C
TCCAGACCCAGCCAGGGAAGGGAATTCAGCTGAAAGCCAGGGCATTCGAACTCTCCGAAG
TGGCAACAACAACTCCGCCCGTTTGCTGGGAGTTTTTAGCCATTCT
TGATGGGGGTGACTTTGAACAGCACATCCAGCAGTCTGTGGCTTGATTTCATCACTGATGCTGA
TCAAATGTGAGGACCCAGGAA
TTTTGCAGGGAGCTCCGTGTCCTTCAGCTGT
CGGCCTCTGCCCACCTGTGTCGCCGAGTGTGGAGGGACAGTGAGAGGAGAGGTGTCGGGGCAGGTGC
TCCAGCTCCCTATGAACACAATCTCAACTGCATCTGGACCATCGAAGCAGAGGCC
TGGGCCTGTGGAGAGCGGGGTTCTGCTGAAGGAGCTGAGTGGCCCGGCCCTGCCCAAGGACCTGC
AGCACCTTCAACTCGGTCGTCCTGCAGTTCAGCACTGACTTCTTCACCAGCAAGCAGGGCTTTGCC
TCAGCTGTGTGAAGATCGAGAACAGGTTCTTCTGGCAGCCCAGCCCGCCAACATGCATC
CTCCCTGCGGGGGAGACCTGACAGGACCATCTGGAGTCATCCTCTCACCAAATTACCCAGAACCCTA
C
TATCTACGACGGACGGGACTCTCTCAGCCCT
TCATAGGAAGCTTCTATGGCTCCCAGCTCCCAGGCCGCATTGAAAGCAGCAGCAACAGCCTCTTCCT
C
GCCTTCCGCAGCGATGCATCTGTGAGCAATGCTGGCTTCGTCATTGACTATACAGAAAACCCGCGGG
TCAAGAACGGCACACGGGTGGGGTCCGACCTGAAGCTGGGCTCC
CCGTCACCTACTACTGCCACGGGGGCTACGAAGTTGAGGGCACCTCGACCCTGAGCTGCATCCTGGG
TCAAGTTCTCATTAAGTTCAGCGCCAAAGGCCTC
CACCAGCCAGAGGCTTCCACTTTGTCTACCAAGCGGTTCCTCGAACCAGCGCCACGCAGTGCAGCTC
TGCAACTCCGGCTATGCCCTGCAGGGGTCGCCAGAGATCGAGTGCCTCCCTGTGCCTGGGGCCTTG
174



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CCCAATGGAATGTCTCAGCGCCCACGTGTGTGGTGCCGTGTGGAGGCAACCTCACAGAGCGCAGGGG
C
CCCCGAAGGCGCTGGCATCCAGATCCAAGTTGTCAGTTTTGTGACAGAGCAGAACTGGGACTCGCTG
TGCTGGGGAGTTTCTCAGGAACAACCGTGCCTGC
CTTCTGAACAGCACCTCCAACCAGCTCTACCTTCATTTCTACTCAGATATCAGCGTATCTGCAGCTG
CTTCCACTTGGAGTACAAAACGGTGGGCCTGAGCAGTTGTCCGGAACCTGCTGTGCCCAGTAACGGG
wU~uAC'ru~cGHGCGC'rHC'1"1'GG'1'G1-~'1'GATGTGGTGTCTTTCCAGTGTGAGCCGGGATATGCCCT
C
CAGGGCCACGCCCACATCTCCTGCATGCCCGGAACAGTGCGGCGATGGAACTACCCTCCTCCACTCT
G
TATTGCACAGTGTGGGGGAACAGTGGAGGAGATGGAGGGGGTGATCCTGAGCCCCGGCTTCCCAGGC
A
ACTACCCCAGTAACATGGACTGCTCCTGGAAAATAGCACTGCCCGTGGGCTTTGGAGCTCACATCCA
G
TTCCTGAACTTCTCCACCGAGCCCAACCACGACTACATAGAAATCCGGAATGGCCCCTATGAGACCA
G
CCGCATGATGGGAAGATTCAGTGGAAGCGAGCTTCCAAGCTCCCTCCTCTCCACGTCCCACGAGACC
A
CCGTGTATTTCCACAGCGACCACTCCCAGAATCGGCCAGGATTCAAGCTGGAGTATCAGGCCTATGA
A
CTTCAAGAGTGCCCAGACCCAGAGCCCTTTGCCAATGGCATTGTGAGGGGAGCTGGCTACAACGTGG
G
TCAATTGACTGGCCACCCTGTCCTCACGTGTCAA
ATGGCACCAACCGGAACTGGGACCACCCCCTGCCCAAGTGTGAAGTCCCTTGTGGCGGGAACATCAC
T
TCTTCCAACGGCACTGTGTACTCCCCGGGGTTCCCTAGCCCGTACTCCAGCTCCCAGGACTGTGTCT
TTGGCCATGGCGTCCGCCTCAACCTCAGCCTGCTGCAGACAGAGCCCTCT
TCACCATCTGGGATGGGCCACAGCAAACAGCACCACGGCTCGGCGTCTTCACCCGGAG
ATGGCCAAGAAAACAGTGCAGAGTTCATCCAACCAGGTCCTGCTCAAGTTCCACCGTGATGCAGCCA
C
CCAACGCCGAAGTCGTCACAGAGAATGAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCT
C
CCTGGCTTTACCTTAGTGGGGAATGAAATTCTGACCTGCAAACTTGGAACCTACCTGCAGTTTGAAG
TC
TTT
TCAGGACAGAGTCCTCTGCTGAAAGCCCTCAGTGGGAATTACTCAGCTCCCCTGATT
TCACGCCTACAATCGGAAGGGCTT
TCCGCTATTCAGCTCTTTCCTGTGGGCTTCCTGAGGCCCCCAAGAATGGAATGGTGTTTGGCA
175



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TGGAGCAACCGCAATGTCCCACCACAGTGTGTCCC
GTGACTTGTCCTGATGTCAGTAGCATCAGCGTGGAGCATGGCCGATGGAGGCTTATCTTTGAGACAC
TCAGTTCCAGGCCCAGCTGATGCTCATCTGTGACCCTGGCTACTACTATACTGGCCAAAGGGTC
TGGAGCCTCGGGGACTCTACGCCCACCTGCCGAATCATCTCCTG
TGGCCAATGGGCTC
T
CGGCATGTCTGTGCGCATCTGCCAGCAGGATCATCACTGGTCGGGCAAGACCCCTTTCTGTCAATTA
C
CTGTGGACACCCAGGCAACCCTGTCAACGGCCTCACTCAGGGTAACCAGTTTAACCTCAACGATGTG
CCTGGGTATATGGCTGAGGGGGCTGCTAGGTCCCAATGCCTGGCCAGCGG
TCATCAACTGTACAGATCCTGGACACCAAGAAAATA
CGCACAGGTTCAGCTTCGGCACCACTGTGTCTTACCGGTGC
TGGCACATGGGACCGTCC
--.. ~-~-.-~-~-n.-. ,..~ i ... i ... i a ir,r,w, lr~u i um a m.l: l
Wlu;~;~:la.W u; LaW;Cil:'1'c.:C: C:C:C;CCTCACTCCCAGAT
G
TCTGGAGACAGTTATACTGTGGGAGCAGTGGTGCGGTACAGCTGCATCGGCAAGCGTACTCTGGTGG
CTCCCTCACTGCTCAGGAACC
TCCCGGCTCATGGCATCCGTTTGGGGGACAGCTTTGATCC
TCTCTTGTGGGAACCCTGGGACT
CAAGTAATGCCCGAGTTGTGTTCAGTGATGGCCTGGTTTTCTCCAGCTCTATCGTCTATGAGTGCCG
TGGTACCTGGACAGGCAGTG
TTCCAGCCAATGGCCTTCGGCTGGGCAAT
TCAGTGTGTCCCTGGCTATATGATGGAGTCACATAGAGT
TCCCCAATGGGAAGGTGGTGGGGTCTGACTTCATGTGGGGCTCAAGTGTG
CTTATGCCTGCCTGGAGGGGTACCAGCTCTCCCTGCCCGCGGTGTTCACCTGTGAGGGAAATGGGTC
C
TCTGTCTCCTTCTCCTGCCATCCCCCTCTGGTGCTG
176



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
A
CCAGGTTGGAAGCACAGTCCTCTTCCGTTGTCAAAAAGGCTACCTGCTTCAGGGCTCCACCACCAGG
A_
CCTGCCTCCCAAACCTGACCTGGAGTGGAACCCCACCTGACTGTGTCCCCCACCACTGCAGGCAGCC
A_
GAGACGCCAACGCATGCCAACGTCGGGGCCCTGGATTTGCCCTCCATGGGCTACACGCTCATTACTC
_C
TGCCAGGAGGGCTTCTCCCTCAAGGGTGGCTCCGAGCACCGCACCTGCAAGGCGGATGGCAGCTGGA
_C
AGGCAAGCCGCCCATCTGCCTGGAGGTCCGGCCCAGTGGGAGACCCATCAACACTGCCCGGGAGCCA
_C
CGCTCACCCAAGCCTTGATTCCTGGGGATGTTTTTGCCAAGAATTCCCTGTGGAAAGGGGCCTATGA
_A
TACCAGGGGAAGAAGCAGCCAGCCATGCTCAGAGTGACTGGCTTCCAAGTTGCCAACAGCAAGGTCA
A_
TGCCACCATGATCGACCACAGTGGCGTGGAGCTGCACTTGGCTGGAACTTACAAGAAAGAAGATTTT
_C
ATCTCCTACTCCAGGTGTACCAGATTACAGGGCCTGTGGAGATCTTTATGAATAAGTTCAAAGATGA
_T
CACTGGGCTTTAGATGGCCATGTCTCGTCAGAGTCCTCCGGAGCCACCTTCATCTACCAAGGCTCTG
_T
CAAGGGCCAAGGCTTTGGGCAGTTCGGCTTTCAAAGACTGGACCTCAGGCTGCTGGAGTCAGACCCC '
_G
AGTCCATTGGCCGCCACTTTGCTTCCAACAGCAGCTCAGTGGCAGCCGCGATCCTGGTGCCTTTCAT '
_C
GCCCTCATTATTGCGGGCTTCGTGCTCTATCTCTACAAGCACAGGAGAAGACCCAAAGTTCCTTTCA I
_A
TGGCTATGCTGGCCACGAGAACACCAATGTTCGGGCCACATTTGAGAACCCAATGTACGACCGCAAC
A j
TCCAGCCCACAGACATCATGGCCAGCGAGGCGGAGTTCACAGTCAGCACAGTGTGCACAGCAGTATA
_G
CCACCCGGCCTGGCCGCTTTTTTTGCTAGGTTGAACTGGTACTCCAGCAGCCGCCGAAGCTGGACTG
_T
ACTGCTGCCATCTCAGCTCACTGCAACCTCCCTGCCTGATTCCCCTGCCTCAGCCTGCCGAGTGCCT
_G
CGATTGCAGGCGCGCACCGCCAC
. ~.~_W:u.~
NOV4d, CG50377-03 SEQ~~ NO: 38 2612 aa~ 3MW at 284047.SkD
Protein Sequence
__
MAGAPPPALLLPCSLISDCCASNQRHSVGVGPSELVKKQIELKSRGVKLMPSKDNSQKTSVLTQVGV
S
QGHNMCPDPGIPERGKRLGSDFRLGSSVQFTCNEGYDLQGSKRITCMKVSDMFAAWSDHRPVCRARM
C
DAHLRGPSGIITSPNFPIQYDNNAHCVWIITALNPSKVIKLAFEEFDLERGYDTLTVGDGGQDGDQK
T
VLYMSQNACSDSPHTPGSRIPESMSGDIWRQKWTVLEICRDISSSDARSGSVRKSPKTSNAVELVAP
G
TEIEQGSCGDPGIPAYGRREGSRFHHGDTLKFECQPAFELVGQKAITCQKNNQWSAKKPGCVFSCFF
N
FTSPSGVVLSPNYPEDYGNHLHCVWLILARPESRIHLAFNDIDVEPQFDFLVIKDGATAEAPVLGTF
S
GNQLPSSITSSGHVARLEFQTDHSTGKRGFNITFTTFRHNECPDPGVPVNGKRFGDSLQLGSSISFL
C
~DEGFLGTQGSETTTCVLKEGSVVWNSAVLRCEAPCGGHLTSPSGTILSPGWPGFYKDALSCAWVIEA
'Q
PGYPIKITFDRFKTEVNYDTLEVRDGRTYSAPLIGVYHGTQVPQFLISTSNYLYLLFSTDKSHSDIG
F
QLRYETITLQSDHCLDPGIPVNGQRHGNDFYVGALVTFSCDSGYTLSDGEPLECEPNFQWSRALPSC
E
ALCGGFIQGSSGTILSPGFPDFYPNNLNCTWIIETSHGKGVFFTFHTFHLESGHDYLLTTENGSFTQ
P
177



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
SDFSMSYEGFNITFSEYDLEPCEEPEVPAYSIRKGLQFG
SPLPRCVAECGNSVTGTQGTLLSPNFPVNYNNNHEC
IQTQPGKGTQLKARAFELSEGDVLKWDGNNNSARLLGVFSHSEMMGVTLNSTSSSLWLDFITDA
SKGFELHFSSFELIKCEDPGTPKFGYKVHDEGHFAGSSVSFSCDPGYSLRGSEELLCLSGERRTW
CGGTVRGEVSGQVLSPGYPAPYEHNLNCIWTIEAEAGCTIGLHFLVFDTEEVHDVLRI
STDFFTSKQGFAIQFSVSTATSCNDPGIPQNGSR
ISCVKIENRFFWQPSPPTCIAPCGGDLTGPSGVILSPNYPEP
PDWIALVFNIFNLEPGYDFLHIYDGRDSLSPLIGSFYGSQLPGRIESSSNSLF
FVIDYTENPRESCFDPGSIKNGTRVGSDLKLGSSVTYYCHGGYEVEGTSTLSCIL
PNYPQNYTSGQICLYFVTVPKDYWFGQFAFFHTAL
LPLATSNQVLIKFSAKGLAPARGFHFWQAVPRTSATQCS
YGKRLGSDFSVGAIVRFECNSGYALQGSPEIECLPVPGALAQWNVSAPTCWPCGGNLTERR
ILSPGFPEPYLNSLNCVWKIWPEGAGIQIQWSFWEQNWDSLEVFDGADNTVTMLGSFSGTTVP
FHLEYKTVGLSSCPEPAVPSNGVKTGERYLVNDWSFQCEPGYA
CMPGTVRRWNYPPPLCIAQCGGTVEEMEGVILSPGFPGNYPSNMDCSWKIALPVGFGAHI
STEPNHDYIEIRNGPYETSRMMGRFSGSELPSSLLSTSHETTWFHSDHSQNRPGFKLEYQAY
PFANGIVRGAGYNVGQSVTFECLPGYQLTGHPVLTCQHGTNRNWDHPLPKCEVPCGGNI
T
SNGTWSPGFPSPYSSSQDCWLITVPIGHGVRLNLSLLQTEPSGDFITIWDGPQQTAPRLGVFTR
SNQVLLKFHRDAATGGIFAIAFSAYPLTKCPPPTILPNAEWTENEEFNIGDIVRYRC
VGNEILTCKLGTYLQFEGPPPICEVHCPTNELLTDSTGVILSQSYPGSYPQFQTCSWLVRVE
SLTVEYFLSEKQYDEFEIFDGPSGQSPLLKALSGNYSAPLIVTSSSNSVYLRWSSDHAYNRKG
F
SCSEGYHLQAGAEATAECLDTGLWSNRNVPPQCV
SVEHGRWRLIFETQYQFQAQLMLICDPGYWTGQRVIRCQANGKWSLGDSTPTCRIIS
PPNGHRIGTLSWGATAIFSCNSGYTLVGSRVRECMANGLWSGSEVRCLAGHCGTPEPIVNG
SYRGSVWQCNAGFRLIGMSVRICQQDHHWSGKTPFCQLPVDTQATLSTASLRVTSLTSTM
SSTVQILDTKKIVFVRSTPAARTGSASAPLCLTG
WAPQCSAAREMAHGTVPAPSVSVSSGVLWPSGLPASLPDWRQLYCGSSGAVQLHRQAYSG
KQHPHVWAGWTLDWLPPSLLRNQRGSLR
4e CG50377 OS SE m NO. 39 10466 b _ ____
. . TAG at 10450
Sequence ORF Startv at 1 ~~~,~ORF St p'
17~



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TGTCCTGTCGGTGTTTGATGGTCCACCCCAGCCAGA
TGCCAGCCACCATTGTTAGTGCAGCCACCACCCTCT
C
C
ACAGCTGCAACCTTGGCTTCTTCCTGGAGGGCCACGCCGTGCTCACCTGCCACG
TCTCCAGCCCCCACTTCCCCTCGGAGTACCATAACAATGC
T
GGAGGATGGTTACGACTTTCTGGAAGTCACTGGGACAGAAGGCTCCTCCCTCTGGTTCACCGGAGCC
A
GCCTCCCAGCCCCCGTTATCAGCAGCAAGAACTGGCTGCGACTGCACTTCACATCGGATGGCAACCA
AACTCAGGTTGGTGTGTCCCAAGGA
TGACCTGCAAGGGTCCAAGCGGATCACCTGTATGA
GGCCCCTCGGGCATCATCACCTCCCCCAATTTCCCCATTCAGTATGACAACAATGCACA
GATCATCACAGCACTCAACCCCTCCAAGGTAATCAAGCTCGCCTTTGAGGAGTTTGATT
GCTATGACACCCTGACGGTCGGTGATGGTGGTCAGGATGGGGACCAGAAGACAGTTCTC
ACAGGTACATCGGTCCCGGATCTCATTGTCAGCACCAATCATCAAATGTGGCTCCTCTT
TGGCAGTGGCAGTTCCCTGGGATTCAAGGCTTCTTATGAAGAGATCGAGCAGGGCAGTT
CTGGCATACCTGCATATGGCCGGAGGGAAGGCTCCCGGTTTCGCCACGGTGACACACTC
TGCCAGCCCGCCTTTGAGCTGGTGGGACAGAAGGCAATCACATGCCAAAAGAATAACCA
CCTGTCTCCCAACTACCCAGAGGACTATGGCAACCACCTCCACTGTGTCTGGCTCATCCTGGCCAGG
C
CTGAGAGCCGCATCCACCTGGCCTTCAACGACATTGACGTGGAGCCTCAGTTTGATTTCCTGGTCAT
C
CACAAGCAGTGGCCACGTGGCCCGTCTCGAGTTCCAGACTGACCACTCCACAGGGAAGAGGGGCTTC
A
ACATCACTTTTACCAGTGAGTCCTCCAACGAGTGCCCGGATCCTGGCGTTCCAGTAAATGGCAAACG
G
TTTGGGGACAGCCTCCAGCTGGGCAGCTCCATCTCCTTCCTCTGTGATGAAGGCTTCCTTGGGACTC
179



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
C
ACCACGGGACCCAGGTTCCCCAGTTCCTCATCAGCACCAGCAACTACCT
C
TACCTCCTCTTCTCTACCGACAAGAGTCACTCGGACATCGGCTTCCAGCTCCGCTATGAGGCTATAA
TCCAGGAATCCCAGTAAATGGACAGCGTCATGGGAATGACTTC
ACACATTAAGTGACGGGGAGCCTCTGGA
TGTGAGCCCAACTTCCAGTGGAGCCGGGCCCTGCCCAGTTGTGAAGCTCTCTGTGGTGGCTTCATTC
TTATCGAAACATCTCATGGCAAGGGTGTGTTCTTCACTTTCCACACCTTCCACCTGGAAAGTGG
C
TCTCCATGTCATATGAAGGATTCAACATCACCTTCTCAGAGTACGACTTGGAGCCCTGTGAGGAGCC
CTTCCCCGGGTACCGTCTGGAGGGCACCGCCCGCATCACGTGCCTGGGGGGCAGACGGCGCCTGTGG
TAACAATCATGAATGCATCTACTCCATCCAGACCCAGCCAG
G
TCAAATGTGAGGACCCAGGAACCCCCAAGTTTGGCTACAAGGTT
C
CGAGTGTGGAGGGACAGTGAGAGGAGAGGTGTCGGGGCAGGTGCTGTCACCCGGGTATCCAGCTCCC
T
TCTGCTGAAGGAGCTGAGTGGCCCGGCCCTGCCCAAGGACCTGCATAGCACCTTCAACTCGGTCGTC
CTCCACAGTGTTCCAGTGTGACCCTGGCTACGCGCTGCAGGGAAGTGCAGAGATCAGCTGTGTGAAG
180



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
GAAAGTGACCGTCTCACCAGACTACGTCATCGCCCTGGTATTGTTCCCCAGCTTTAACCTGGAGCCT
ATGACTTCCTCCATATCTACGACGGACGGGACTCTCTCAGCCCTCTCATAGGAAGCTTCTATGG
CCAGCTCCCAGGCCGCATTGAAAGCAGCAGCAACAGCCTCTTCCTCGCCTTCCGCAGCGATGCAT
TTGACTTCCCAGAAAACCCGCGGGAGTCATGTTTTGATCCTGGT
CCATCAAGAACGGCACACGGGTGGGGTCCGACCTGAAGCTGGGCTCCTCCGTCACCTACTACTGCCA
GGGGGCTACGAAGTTGAGGGCACCTCGACCCTGAGCTGCATCCTGGGGCCTGATGGGAAGCCCGTGT
ACCCCCAGAACTACACCAGTGGACAGATCTGCTTGTATTTTGTTACTGTGCCCAA
TGGTGTGGTGTTTGGCCAGTTCGCCTTCTTTCACACGGCCCTCAACGACGTGGTGGAGGTTC
CGACGGCCACAGCCAGCACTCGCGGCTCCTCAGCTCCCTCTCGGGCTCCCATACAGGTGAATCACTG
CCTTGGCCACCTCCAATCAAGTTCTCATTAAGTTCAGCGCCAAAGGCCTCGCACCAGCCAGAGGCTT
CACTTTGTCTACCAAGCGGTTCCTCGAACCAGCGCCACGCAGTGCAGCTCTGTGCCGGAACCCCGCT
TCGAGTGCCTCCCTGTGCCTGGGGCCTTGGCCCAATGGAATGTCTC
CTTCCCAGAGCCGTACCTCAACAGCCTCAACTGTGTGTGGAAGATCGTGGTCCCCGAAGGCGCTGGC
TCCAGATCCAAGTTGTCAGTTTTGTGACAGAGCAGAACTGGGACTCGCTGGAAGTATTTGATGGTGC
GATAACACTGTAACCATGCTGGGGAGTTTCTCAGGAACAACCGTGCCTGCCCTTCTGAACAGCACCT
TATCAGCGTATCTGCAGCTGGCTTCCACTTGGAGTAC
ACTTGGTGAATGATGTGGTGTCTTTCCAGTGTGAGCCGGGATATGCCCTCCAGGGCCACGCCCACA
TGCCCGGAACAGTGCGGCGATGGAACTACCCTCCTCCACTCTGTATTGCACAGTGTGGG
ACCCCAGTAACAT
GACTGCTCCTGGAAAATAGCACTGCCCGTGGGCTTTGGAGCTCACATCCAGTTCCTGAACTTCTCCA
AGAAATCCGGAATGGCCCCTATGAGACCAGCCGCATGATGGGAAGA
ATTTCCACAG
ATCAGGCCTATGAACTTCAAGAGTGCCCAG
CCAGAGCCCTTTGCCAATGGCATTGTGAGGGGAGCTGGCTACAACGTGGGACAATCAGTGACCTTC
181



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
ATCAATTGACTGGCCACCCTGTCCTCACGTGTCAACATGGCACCAACCGGAA
T
GTACTCCCCGGGGTTCCCTAGCCCGTACTCCAGCTCCCAGGACTGTGTCTGGCTGATCACCGTGCCC
C
AGCTTTCTCCGCTTATCCACTCACCAAATGCCCTCCTCCCACCATCCTCCCCAACGCCGAAGTCGT
TGAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCTCCCTGGCTTTACCTTAG
GGAAGCTATCCCCAGTTCCAGACCTGCTCTTGGCTGGTGAGAGTGGAGCCCGACTATAACATCTCCC
T
CACAGTGGAGTACTTCCTCAGCGAGAAGCAATATGATGAGTTTGAGATTTTTGATGGTCCATCAGGA
C
C
TTGTCACCAGCTCAAGCAA
CCATGTACAGCTGCAGTGAAGGCTACCACCTCCAGGCAGGCGCTGAGGCCACTGCAGAGTG
TG
C
TGGAGCCTCGGGGACTCTACGCCCACCTGCCGAATCATCTCCTGTGGAGAGCTCCCGATTC
TGGCCAATGGGCTCTGGAGTGGCTCTGAAGT
TTGTCAACGGACACATCAATGGGGAGAACT
CAGCTACCGGGGCAGTGTGGTGTACCAATGCAATGCTGGCTTCCGCCTGATCGGCATGTCTGTGCGC
TCTGCCAGCAGGATCATCACTGGTCGGGCAAGACCCCTTTCTGTGTGCCAATTACCTGTGGACACCC
AACCTCAACGATGTGGTCAAGTTTGTTT
TCAACTGTACAGATCCTGGACACCAAGAAAATAGTGTTCGTCAGGT
CAGTGT
Ig~



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TGGCCTGGTTTTCTCCAGCTCTATCGTCTATGAGTGCCGGGAAGGATACTACGCCACAGG
TGGCCTTCGGCTGGGCAATGACTTCAGGTACAACAAAACT
TATCAGTGTGTCCCTGGCTATATGATGGAGTCACATAGAGTATCTGTGCTGAGCTGCACCAA
C
CAATGGGAAGGTGGTGGGGTCTGACTTCATGTGGGGCTCAAGTGTGACTTATGCCTGCCTGGAGGGG
CCGCGGTGTTCACCTGTGAGGGAAATGGGTCCTGGACCGGAGAGCTGCCTCA
C
CTACAGGTCATCTGTCTCCTTCTCCTGCCATCCCCCTCTGGTGCTGGTGGGCTCTCCACGCAGGTTT
CTTCCGTTGTCAAAAAGGCTACCTGCTTCAGGGCTCCACCACCAGGACCTGCCTCCCAAACCTGACC
C
TCTACTCCTGCCAGGAGGGCTTCTCCC
CAAGGGTGGCTCCGAGCACCGCACCTGCAAGGCGGATGGCAGCTGGACAGGCAAGCCGCCCATCTGC
C
T
CCTGGGGATGTTTTTGCCAAGAATTCCCTGTGGAAAGGGGCCTATGAATACCAGGGGAAGAAGCAGC
C
TGCCACCATGATCGACCAC
C
CAGATTACAGGGCCTGTGGAGATCTTTATGAATAAGTTCAAAGATGATCACTGGGCTTTAGATGGCC
C
TTGGCCGCCACTT
TTATTGCGGGCT
CGTGCTCTATCTCTACAAGCACAGGAGAAGACCCAAAGTTCCTTTCAATGGCTATGCTGGCCACGAG
183



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
~10V4e, CG50377-OS ~SEQ m NO: 40 3483 as ~MW at 379077.1kD
Protein Sequence , .. . -
~QNCTFQLHGPNGTVESPGFPYGYPNYANCTWTITAEEQHRIQLVFQSFALEEDFDVLSVFDGPPQP
E
ULRTRLTGFQLPATIVSAATTLSLRLISDYAVSAQGFHATYEVLPSHTCGNPGRLPNGIQQGSTFNL
J
DKVRYSCNLGFFLEGHAVLTCHAGSENSATWDFPLPSCRADDACGGTLRGQSGIISSPHFPSEYHNN
A
DCTWTILAELGDTIALVFIDFQLEDGYDFLEVTGTEGSSLWFTGASLPAPVISSKNWLRLHFTSDGN
H
RQRGFSAQYQVKKQIELKSRGVKLMPSKDNSQKTSVVTQVGVSQGHNMCPDPGIPERGKRLGSDFRL
G
SSVQFTCNEGYDLQGSKRITCMKVSDMFAAWSDHRPVCRARMCDAHLRGPSGIITSPNFPIQYDNNA
H
CVWIITALNPSKVIKLAFEEFDLERGYDTLTVGDGGQDGDQKTVLYMLTGTSVPDLIVSTNHQMWLL
~TDGSGSSLGFKASYEEIEQGSCGDPGIPAYGRREGSRFRHGDTLKFECQPAFELVGQKAITCQKNN
Q
WSAKKPGCVCSCFFNFTSPSGVVLSPNYPEDYGNHLHCVWLILARPESRIHLAFNDIDVEPQFDFLV
I
KDGATAEAPVLGTFSGNQLPSSITSSGHVARLEFQTDHSTGKRGFNITFTSESSNECPDPGVPVNGK
R
FGDSLQLGSSISFLCDEGFLGTQGSETITCVLKEGSVVWNSAVLRCEAPCGGHLTSPSGTILSPGWP
G
FYKDALSCAWVIEAQPGYPIKITFDRFKTEVNYDTLEVRDGRTYSAPLIGVYHGTQVPQFLISTSNY
LFSTDKSHSDIGFQLRYEAITLQSDHCLDPGIPVNGQRHGNDFYVGALVTFSCDSGYTLSDGEPL
PNFQWSRALPSCEALCGGFIQGSSGTILSPGFPDFYPNNLNCTWIIETSHGKGVFFTFHTFHLES
YLLITENGSFTQPLRQLTGSRLPAPISAGLYGNFTAQVRFISDFSMSYEGFNITFSEYDLEPCEE
PAYSIRKGLQFGVGDTLTFSCFPGYRLEGTARITCLGGRRRLWSSPLPRCVAECGNSVTGTQGTL
NFPVNYNNNHECIYSIQTQPGKGIQLKARAFELSEGDVLKVYDGNNNSARLLGVFSHSEMMGVTL
STSSSLWLDFITDAENTSKGFELHFSSFELIKCEDPGTPKFGYKVHDEGHFAGSSVSFSCDPGYSLR
G
SEELLCLSGERRTWDRPLPTCVAECGGTVRGEVSGQVLSPGYPAPYEHNLNCIWTIEAEAGCTIGLH
F
LVFDTEEVHDVLRIWDGPVESGVLLKELSGPALPKDLHSTFNSWLQFSTDFFTSKQGFAIQFSGST
A
TSCNDPGIPQNGSRSGDSWEAGDSTVFQCDPGYALQGSAEISCVKIENRFFWQPSPPTCIAPCGGDL
PNYPEPYPPGKECDWKVTVSPDYVIALVLFPSFNLEPGYDFLHIYDGRDSLSPLIGSFY
SSSNSLFLAFRSDASVSNAGFVIDFPENPRESCFDPGSIKNGTRVGSDLKLGSSVTYYC
STLSCILGPDGKPVWNNPRPVCTAPCGGQYVGSDGVVLSPNYPQNYTSGQICLYFVTVP
SLSGSHTGESLPLATSNQVLIKFSAKGLAPARG
VPRTSATQCSSVPEPRYGKRLGSDFSVGAIVRFECNSGYALQGSPEIECLPVPGALAQWNV
PCGGNLTERRGTILSPGFPEPYLNSLNCVWKIWPEGAGIQIQVVSFVTEQNWDSLEVFDG
LGSFSGTTVPALLNSTSNQLYLHFYSDISVSAAGFHLEYKTVGLSSCPEPAVPSNGVKTGE
184



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
VNDVVSFQCEPGYALQGHAHISCMPGTVRRWNYPPPLCIAQCGGTVEEMEGVILSPGFPGNYPSN
ELPSSLLSTSHETTVYFH
CPDPEPFANGIVRGAGYNVGQSVTFECLPGYQLTGHPVLTCQHGTNR
SNGTVYSPGFPSPYSSSQDCVWLITVPIGHGVRLNLSLLQTEPSGDFIT
IFAIAFSAYPLTKCPPPTILPNAEV
GDIVRYRCLPGFTLVGNEILTCKLGTYLQFEGPPPICEVHCPTNELLTDSTGVILSQSY
SLTVEYFLSEKQYDEFEIFDGPSGQSPLLKALSGNYSAPLIVTSSS
SVYLRWSSDHAYNRKGFKIRYSALSCGLPEAPKNGMVFGKEYTVGTKAMYSCSEGYHLQAGAEATAE
WSNRNVPPQCVPVTCPDVSSISVEHGRWRLIFETQYQFQAQLMLICDPGYYYTGQRVIRCQA
GKWSLGDSTPTCRIISCGELPIPPNGHRIGTLSVYGATAIFSCNSGYTLVGSRVRECMANGLWSGSE
IVNGHINGENYSYRGSVVYQCNAGFRLIGMSVRICQQDHHWSGKTPFCVPITCGH
P -
GNPVNGLTQGNQFNLNDVVKFVCNPGYMAEGAARSQCLASGQWSDMLPTCRIINCTDPGHQENSVRQ
FGTTVSYRCNHGFYLLGTPVLSCQGDGTWDRPRPQCLLVSCGHPGSPPHSQMSGDSYT
YSCIGKRTLVGNSTRMCGLDGHWTGSLPHCSGTSVGVCGDPGIPAHGIRLGDSFDPGTVMRF
WSGSQPECGVISCGNPGTPSNARWFSDGLVFSSSIVYECREGYYAT
VINCGDPGIPANGLRLGNDFRYNKTVTYQCVPGYMMESHRVSVLSCT
LIPNGKWGSDFMWGSSVTYACLEGYQLSLPAVFTCEGNGSWTGELP
.GFSYRSSVSFSCHPPLVLVGSPRRFCQSDGTWSGTQPSCIDPTLTTC
IQNNSQGYQVGSTVLFRCQKGYLLQGSTTRTCLPNLTWSGTPPDCVPHHCRQPETPTHA
IYSCQEGFSLKGGSEHRTCKADGSWTGKPPICLEVRPNGRPINTAREPPLTQAL
AKNSLWKGAYEYQGKKQPAMLRVTGFQVANSKVNATMIDHSGVELHLAGTYKKEDFHLLLQV
TGPVEIFMNKFKDDHWALDGHVSSESSGATFIYQGSVKGQGFGQFGFQRLDLRLLESDPESIGRH
SVAAAILVPFIALIIAGFVLYLYKHRRRPKVPFNGYAGHENTNVRATFENPMYDRNIQPTDI
EAEFTVSTVCTAV
4f, CG50377-06 SE_Q m NO: 41 12901 by _ _ _
Sequence _ ORF Start: ATG at 889 ORF Stop: TAG at 102_79
T rT T rmr_ra rrxmmt'~rAA(''TCtC'_ACGGTCCCAATGGGACAGTTGAGAGCCCAGGGTTCCCATATG
CAATTACGCCAACTGCACGTGGACCATCACCGCGGAAGAGCAGCACAGAATCCAGCTTGTG
CACCCTCT
TGAAGTTCTCCCC
185



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
A
GCCACACATGTGGGAACCCAGGGAGGCTGCCCAATGGCATCCAGCAGGGTTCAACCTTCAACCTCGG
T
GACAAGGTCCGCTACAGCTGCAACCTTGGCTTCTTCCTGGAGGGCCACGCCGTGCTCACCTGCCACG
C
TGGCTCTGAGAACAGCGCCACGTGGGACTTCCCCCTGCCTTCCTGCAGAGCTGATGATGCCTGTGGT
G
GGACCCTGCGGGGCCAGAGTGGCATCATCTCCAGCCCCCACTTCCCCTCGGAGTACCATAACAATGC
C
GACTGCACATGGACCATCCTGGCTGAGCTGGGGGACACCATCGCCCTGGTGTTTATTGACTTCCAGC
T
GGAGGATGGTTACGACTTTCTGGAAGTCACTGGGACAGAAGGCTCCTCCCTCTGGTTCACCGGAGCC
A
GCCTCCCAGCCCCCGTTATCAGCAGCAAGAACTGGCTGCGACTGCACTTCACATCGGATGGCAACCA
C
'CGGCAGCGCGGATTCAGTGCCCAATACCAAGTCAAGAAGCAAATTGAGTTGAAGTCTCGAGGTGTGA
A
GCTGATGCCCAGCAAAGACAACAGCCAGAAGACGTCTGTGGTAACTCAGGTTGGTGTGTCCCAAGGA
C
ATAATATGTGTCCAGACCCTGGCATACCCGAAAGGGGCAAAAGACTAGGCTCGGATTTCAGGTTAGG
A
TCCAGCGTCCAGTTCACCTGCAACGAGGGCTATGACCTGCAAGGGTCCAAGCGGATCACCTGTATGP
A
AGTGAGCGACATGTTTGCGGCCTGGAGCGACCACAGGCCAGTCTGCCGAGCCCGCATGTGTGATGCC
C
ACCTTCGAGGCCCCTCGGGCATCATCACCTCCCCCAATTTCCCCATTCAGTATGACAACAATGCACF
C '
TGTGTGTGGATCATCACAGCACTCAACCCCTCCAAGGTAATCAAGCTCGCCTTTGAGGAGTTTGAT7
T
GGAGAGGGGCTATGACACCCTGACGGTCGGTGATGGTGGTCAGGATGGGGACCAGAAGACAGTTCTC
T
ACATGCTGACAGGTACATCGGTCCCGGATCTCATTGTCAGCACCAATCATCAAATGTGGCTCCTCT'.
C
CAGACTGATGGCAGTGGCAGTTCCCTGGGATTCAAGGCTTCTTATGAAGAGATCGAGCAGGGCAGT'.
G
CGGTGACCCTGGCATACCTGCATATGGCCGGAGGGAAGGCTCCCGGTTTCGCCACGGTGACACACT~
A
AGTTTGAGTGCCAGCCCGCCTTTGAGCTGGTGGGACAGAAGGCAATCACATGCCAAAAGAATAACC~
A
TGGTCGGCTAAGAAGCCAGGCTGCGTGTGTTCCTGCTTCTTCAACTTCACCAGCCCGTCTGGGGTT~
CACCTCCACTGTGTCTGGCTCATCCTGGCCAGG
.TTTCCTGGTCAT
.TGGGGCCACCGCCGAGGCGCCCGTCCTGGGCACCTTCTCAGGAAACCAGCTTCCCTCCTCCA
TCACTTTTACCAGTGAGTCCTCCAACGAGTGCCCGGATCCTGGCGTTCCAGTAAATGGCAAACG
TCACCTGCGTCCTGAAGGAGGGCAGCGTGGTCTGGAACAGCGCTGTGCTGCGG
CCTGTGGTGGTCACCTGACTTCGCCCAGCGGCACCATCCTCTCTCCGGGCTGGCCTGG
186



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
ACCACGGGACCCAGGTTCCCCAGTTCCTCATCAGCACCAGCAACTACCT
TCCAGGAATCCCAGTAAATGGACAGCGTCATGGGAATGACTTC
T
TGTGAGCCCAACTTCCAGTGGAGCCGGGCCCTGCCCAGTTGTGAAGCTCTCTGTGGTGGCTTCATTC
A
AGGCTCCAGTGGGACCATCTTGTCGCCAGGTTTCCCTGACTTCTACCCCAACAACTTGAACTGCACC
T
GGATTATCGAAACATCTCATGGCAAGGGTGTGTTCTTCACTTTCCACACCTTCCACCTGGAAAGTGG
e-iv~.1 ~.~.wrwra~~~wr~~~wrwrH'rUCica~AC'1"1'CAC't'GCCCAGGTCCGCTTCATCTCTGAT
TGTCATATGAAGGATTCAACATCACCTTCTCAGAGTACGACTTGGAGCCCTGTGAGGAGCC
r~~~~~U~'t'AC:CCs't'c:'t'UUAU~UC:AC:CGCCCGCATCACGTGCCTGGGGGGCAGACGGCGCCTGTGG
TCGCCTCTGCCAAGGTGTGTTGCTGAGTGTGGGAATTCAGTCACAGGCACTCAGGGTACTTTGCT
CCCCAACTTTCCTGTGAACTACAATAACAATCATGAATGCATCTACTCCATCCAGACCCAGCCAG
GAAGGGAATTCAGCTGAAAGCCAGGGCATTCGAACTCTCCGAAGGAGATGTCCTCAAGGTTTATGAT
G
GCAACAACAACTCCGCCCGTTTGCTGGGAGTTTTTAGCCATTCTGAGATGATGGGGGTGACTTTGAA
C
AGCACATCCAGCAGTCTGTGGCTTGATTTCATCACTGATGCTGAAAACACCAGCAAGGGCTTTGAAC
C
c:C:'t"1 c:AGCTGTGACCCTGGATACAGCCTGCGGGG
CGGACCTGGGACCGGCCTCTGCCCACCTGTGTCG
GGGGCAGGTGCTGTCACCCGGGTATCCAGCTCCC
AAGCAGAGGCCGGCTGCACCATTGGGCTACACTT
CTGCGCATCTGGGATGGGCCTGTGGAGAGCGGGG
CAAGGACCTGCATAGCACCTTCAACTCGGTCGTC
C
ACGTCCTGCAATGACCCTGGGATCCCGCAGAATGGGAGTCGGAGTGGTGACAGTTGGGAAGCCGGCG
A
CTCCACAGTGTTCCAGTGTGACCCTGGCTACGCGCTGCAGGGAAGTGCAGAGATCAGCTGTGTGAAG
TTGTTCCCCAGCTTTAACCTGGAGCCT
187



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
GCTATGACTTCCTCCATATCTACGACGGACGGGACTCTCTCAGCCCTCTCATAGGAAGCTTCTATGG
C
TCCCAGCTCCCAGGCCGCATTGAAAGCAGCAGCAACAGCCTCTTCCTCGCCTTCCGCAGCGATGCAT
C
TGTGAGCAATGCTGGCTTCGTCATTGACTTCCCAGAAAACCCGCGGGAGTCATGTTTTGATCCTGGT
CCATCAAGAACGGCACACGGGTGGGGTCCGACCTGAAGCTGGGCTCCTCCGTCACCTACTACTGCCA
C
TGGGAAGCCCGTGT
CAGTCTGCACAGCCCCCTGTGGGGGACAGTATGTGGGTTCGGACGGAGTGGTC
TCTGCTTGTATTTTGTTACTGTGCCCAA
CCTCTCGGGCTCCCATACAGGTGGAGAATCA
TTAAGTTCAGCGCCAAAGGCCTCGCACCAGCCAGAGG
T
C
TCCTGTCCC
C
TCCAAGTTGTCAGTTTTGTGACAGAGCAGAACTGGGACTCGCTGGAAGTATTTGATGG
TGCTGGGGAGTTTCTCAGGAACAACCGTGCCTGCCCTTCTGAACAGCA
TTTCTACTCAGATATCAGCGTATCTGCAGCTGGCTTCCACTTGGAG
TGATGTGGTGTCTTTCCAGTGTGAGCCGGGATATGCCCTCCAGGGCCACGCCC
CATCTCCTGCATGCCCGGAACAGTGCGGCGATGGAACTACCCTCCTCCACTCTGTATTGCACAGTGT
TGGAGGGGGTGATCCTGAGCCCCGGCTTCCCAGGCAACTACCCCAGTAA
C
TGGACTGCTCCTGGAAAATAGCACTGCCCGTGGGCTTTGGAGCTCACATCCAGTTCCTGAACTTCT
TCCGGAATGGCCCCTATGAGACCAGCCGCATGATGGGA
TTCAGTGGAAGCGAGCTTCCAAGCTCCCTCCTCTCCACGTCCCACGAGACCACCGTGTATTTCCA
TTCAAGCTGGAGTATCAGGCCTATGAACTTCAAGAGTGCC
riven:~.~:rivriti~:m:w-
mmt.wtwut;A't°t'G'1'GAGGGGAGCTGGCTACAACGTGGGACAATCAGTGACC
T
TCGAGTGCCTCCCGGGGTATCAATTGACTGGCCACCCTGTCCTCACGTGTCAACATGGCACCAACCG
G
C
TTGGCCATGGCGTCCGCCTCAACCTCAGCCTGCTGCAGACAGAGCCCTCTGGAGATTTCATCAC
18g



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
C
C
TGCAGCCACAGGGGGGATCTTC
TAGCTTTCTCCGCTTATCCACTCACCAAATGCCCTCCTCCCACCATCCTCCCCAACGCCGAAGT
TGAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCTCCCTGGCTTTACCT
TGAAATTCTGACCTGCAAACTTGGAACCTACCTGCAGTTTGAAGGACCACCCCCGATA
.TGAGCTTCTGACAGACTCCACAGGCGTGATCCTGAGCCAGAGCTA
TAACATCT
~wrw~~~~ruc~~wrHwr~rcc~t'cAGCGAGAAGCAATATGATGAGTTTGAGATTTTTGATGGTCCATCA
G
GACAGAGTCCTCTGCTGAAAGCCCTCAGTGGGAATTACTCAGCTCCCCTGATTGTCACCAGCTCAAG
C
C
TGGCTTCATCCTAGGCCAGACCAGCACCCAG
TGGCCATCTG
TCCCTCTCTGTCAAGCTCTTTCCTGTG
TGGTGTTTGGCAAGGAGTACACAGTGGGAACCAAGGCCATG
ACCACCTCCAGGCAGGCGCTGAGGCCACTGCAGAGTGTCTGGACACAGG
CTATGGAGCAACCGCAATGTCCCACCACAGTGTGTCCCTGTGACTTGTCCTGATGTCAGTAGCATCA
G
CGTGGAGCATGGCCGATGGAGGCTTATCTTTGAGACACAGTATCAGTTCCAGGCCCAGCTGATGCTC
TGGCC
TGGCCAATGGGCTCTGGAGTGGCTCTGAAGTCCGCTGCCTTGC
TGGGGAGAACTACAGCTACCGGG
CAGTGTGGTGTACCAATGCAATGCTGGCTTCCGCCTGATCGGCATGTCTGTGCGCATCTGCCAGCAG
TCATCACTGGTCGGGCAAGACCCCTTTCTGTGTGCTGGTGTCCTGTGGCCATCCGGGCTCCCCGCC
CACTCCCAGATGTCTGGAGACAGTTATACTGTGGGAGCAGTGGTGCGGTACAGCTGCATCGGCAAGC
TGTGTGGGCTGGATGGACACTGGACTGGCTCCCTCCCTCAC
TGGCATCCGTTTGGGGGA
TGGCTCGTGGAGCGGCTCGCAGCCTGAGTGTGGAGTGATCTCTTGTGGG
TGGCCTGGTTTTCTCCAGCTCTATCGT
189



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
TGGTACCT
TTCCAGCCAATGGCCTT
TGACTTCAGGTACAACAAAACTGTGACATATCAGTGTGTCCCTGGCTATATGATGGA
TAGAGTATCTGTGCTGAGCTGCACCAAGGACCGGACATGGAATGGAACCAAGCCCGTCTGCA
TGCCTGCCTGGAGGGGTACCAGCTCTCCCTGCCCGCGGTGTTCACCTGTGA
TACAGAACA
TGGGCTACA
CAGGAGGGCTTCTCCCTCAAGGGTGGCTCCGAGCACCGCACCTGCAAGGCG
TTCCCTGTGGA
TCGACCACAGTGGCGTGGAGCTGCACTTGGCTGGAACTTACAA
GTTCAAAGATGATCACTGGGCTTTAGATGGCCATGTCTCGTCAGAGTCCTCCGGAGCCACCTTCATC
CACAGCAGTATAGCCACCCGGCCTGGCCGCCACCACCGCCACCGAGAGCCCCGCCTCCAGCAGCCGT
TTTAAAATAGCAAAGCAACTTGTGGATCTGTGTGTGTGTGTGTGTGTGTGTGTATGGGCCTGATCAC.
190



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CTGATGAACGGCCCATCTGCACAGCGGCAGCAAGGATTACCCCAAGGGCAGGAAAAAAGTCTTTGGA
.TTGAAAATCAGTTTCTTTATTTTTTTCAAACACTGTTTTATTGAGTTCTAAACCCAGTGGAT
ATTCTCTGTGCCTGACACTTTACCTATAGTAT
AACACCAAAATGTAGGATTTGTTATTCCCCTTTGACAGATGAGAAAACAGAA
TGA
ACAGTAGCACTGTGGCCAGGTCTGGAAAGCCAGGCAAGACACCTGTGTGTCCTATCTGGGTT
CTCCTTGGCTCCCAGCAGCCGCTGACCATAGCCAGCAAAAGACCTGGCAGGTCCTGAATCAAAACAA
C
ATGCTTGGGTCGTTCATGAATTCCATGAGAATTGTTGGGAAGTTCATGAGTTTAGGAATCAAAGTAT
A
GCATTTAGGGCCACTTTCTCTGTCCTCGGGAAGGCCCCCACAAGCTTAAGCCCCTGTATACGTGTGA
_C
ATGCTGGCCTCAGAGATTCATGTCACATATTGAAATAGCAAATAGAAATGTGCAGGAGCGGAGGGAC
A
GCGACACCTGGTTCTTAAGCAAATTCTAGATTGGAACAATTAGAAGATCTGACTGATGTTAAGAGTG
G
CCAGAGGAAAGTGAGAGGCGTCCATTTCCAGCAGACACAATGGAGGCTGACAGAGGCAGGAGCCTAC
C
CCTGGCCACACCACCTCTGGGACACGGCACCCCCCTGGGCCTGGAGGGAGAGGGCCAATCCAGACAC
A_
CTCACACTCTCATCTCTTTCCTTGGCTGGAAACAAGTGCAGGAAGCTCCCCTCCCAATTTCTTATGC
T
CCCTTCCCCCTTACACTCCTCTCCTTCGACTGCTCCCTTCCCCCTTCTTATGCCCCTCTCAGACCAC
C
CAGCGGATGCCTATTCCCGCCCCCCACCCCTCCCCTGCGTCTCCTTGTAAAGTTCCATGAGATGCTC
_G
AGGAGCACCTGCTGGTGGGGCACCCAGCACTCTGCTGGGCATCACCCTCCGCACCCCTGACATTTCT
C
TCCACCTCCAGAGGCTTGGAAGGGAACAGGGCAGATGAGGCCCTTCACCCACCATCTACGCCATATA
_C
CCATCACCATCCCATGAGG GTAAAACAAAGACP~AAAAA:4TCTTTTGTACAGAGA
T
ATATTTTTATTATACAAAGTTTGTACAGTACCAAAAAAGF~~AP-1AGGTGTAAATTTTTTTTTC
A
191



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
AGGTAAACGGTAGTGGAAGCCTTTTTTGTAAATGTAATAAAATGTATAAATATGGTTTTC
AAATATGTAATCTACACACCTTCTTGGCTTGTCTGCAGTATATACATTT
ATCTGTCTCTGTATATGAGGCTTCCCCCTGGGGTCCTGCATTATGGTACTTTCCTGAATTTGA
TTTTTTGAATTCAAATAAAATATAAATTTTTGCATTG
4f, CG50377-06 SEQ m NO: 42 3130 as MW at 340344.SkD
QKTSWTQVGVSQGHNMCPDPGIPERGKRLGSDFRLGSSVQFTCNEGYDLQGSKRITCMK
SDMFAAWSDHRPVCRARMCDAHLRGPSGIITSPNFPIQYDNNAHCVWITTALNPSKVIK.LAFEEFDL
E
G
YMLTGTSVPDLIVSTNHQMWLLFQTDGSGSSLGFKASYEEIEQGSC
PAYGRREGSRFRHGDTLKFECQPAFELVGQKAITCQKNNQWSAKKPGCVCSCFFNFTSPSGW
IDVEPQFDFLVIKDGATAEAPVLGTFSGNQLPSSI
FNITFTSESSNECPDPGVPVNGKRFGDSLQLGSSISFLCDEGFLGTQ
ITCVLKEGSWWNSAVLRCEAPCGGHLTSPSGTILSPGWPGFYKDALSCAWVIEAQPGYPIKIT
STDKSHSDIGFQLRYEAIT
QSDHCLDPGIPVNGQRHGNDFYVGALVTFSCDSGYTLSDGEPLECEPNFQWSRALPSCEALCGGFIQ
G
SSGTILSPGFPDFYPNNLNCTWIIETSHGKGVFFTFHTFHLESGHDYLLITENGSFTQPLRQLTGSR
L
PAPISAGLYGNFTAQVRFISDFSMSYEGFNITFSEYDLEPCEEPEVPAYSIRKGLQFGVGDTLTFSC
F
PGYRLEGTARITCLGGRRRLWSSPLPRCVAECGNSVTGTQGTLLSPNFPVNYNNNHECIYSTQTQPG
K
GIQLKARAFELSEGDVLKWDGNNNSARLLGVFSHSEMMGVTLNSTSSSLWLDFITDAENTSKGFEL
H
FSSFELIKCEDPGTPKFGYKVHDEGHFAGSSVSFSCDPGYSLRGSEELLCLSGERRTWDRPLPTCVA
E
GLHFLVFDTEEVHDVLRIWDGPVESGV
GPALPKDLHSTFNSWLQFSTDFFTSKQGFAIQFSGSTATSCNDPGIPQNGSRSGDSWEAGD
ISCVKIENRFFWQPSPPTCIAPCGGDLTGPSGVILSPNYPEPYPPGKECDW
PDYVIALVLFPSFNLEPGYDFLHIYDGRDSLSPLIGSFYGSQLPGRIESSSNSLFLAFRSDAS
IKNGTRVGSDLKLGSSVTYYCHGGYEVEGTSTLSCILGPDGKPVW
PNYPQNYTSGQICLYFVTVPKDYGWFGQFAFFHTALNDWEVH
SLSGSHTGGESLPLATSNQVLIKFSAKGLAPARGFHFVYQAVPRTSATQCSSVPEPR
SVGAIVRFECNSGYALQGSPETECLPVPGALAQWNVSAPTCWPCGGNLTERRGTILSP
QIQWSFVTEQNWDSLEVFDGADNTVTMLGSFSGTTVPALLNST
NQLYLHFYSDISVSAAGFHLEYKTVGLSSCPEPAVPSNGVKTGERYLVNDWSFQCEPGYALQGHAH
I
SCMPGTVRRWNYPPPLCIAQCGGTVEEMEGVILSPGFPGNYPSNMDCSWKIALPVGFGAHIQFLNFS
SSLLSTSHETTWFHSDHSQNRPGFKLEYQAYELQECP
192



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
FPSPYSSSQDCVWLITVPIGHGVRLNLSLLQTEPSGDFITIWDGPQQTAPRLGVFTRSMAHICT
QSSSNQVLLKFHRDAATGGIFAIAFSAYPLTKCPPPTILPNAEVVTENEEFNIGDIVRYRCLPGFTL
PPPICEVHCPTNELLTDSTGVILSQSYPGSYPQFQTCSWLVRVEPDYNIS
IFDGPSGQSPLLKALSGNYSAPLIVTSSSNSVYLRWSSDHAYNRKGFKIRYS
LPRAPLHGFILGQTSTQPGGSIHFGCNAGYRLVGHSMAICTRHPQGYHLWSEAIPLCQALSCG
CSEGYHLQAGAEATAECLDTGLWSNRNVPPQCVPVTCPDVSSIS
EHGRWRLIFETQYQFQAQLMLICDPGYYYTGQRVIRCQANGKWSLGDSTPTCRIISCGELPIPPNGH
=GTLSVYGATAIFSCNSGYTLVGSRVRECMANGLWSGSEVRCLAGHCGTPEPIVNGHINGENYSYRG
GMSVRICQQDHHWSGKTPFCVLVSCGHPGSPPHSQMSGDSYTVGAVVRYSCIGKR
~VGNSTRMCGLDGHWTGSLPHCSGTSVGVCGDPGIPAHGIRLGDSFDPGTVMRFSCEAGHVLRGSSE
VFSSSIVYECREGYYATGLLSRHCSVNGTW
PANGLRLGNDFRYNKTVTYQCVPGYMMESHRVSVLSCTKDRTWNGTKPVCK
IPNGKVVGSDFMWGSSVTYACLEGYQLSLPAVFTCEGNGSWTGELPQCFPVFCGDPGVP
SYRSSVSFSCHPPLVLVGSPRRFCQSDGTWSGTQPSCIDPTLTTCADPGVPQFGIQNN
TCLPNLTWSGTPPDCVPHHCRQPETPTHANVGALDLPSMGYT
LKGGSEHRTCKADGSWTGKPPICLEVRPNGRPINTAREPPLTQALIPGDVFAKNSLWK
PAMLRVTGFQVANSKVNATMIDHSGVELHLAGTYKKEDFHLLLQVYQITGPVEIFMNK
SESSGATFIYQGSVKGQGFGQFGFQRLDLRLLESDPESIGRHFASNSSSVAAAIL
IAGFVLYLYKHRRRPKVPFNGYAGHENTNVRATFENPMYDRNIQPTDIMASEAEFTVSTVC
4g, 273095147 ~SEQ_m NO: 43 2329 by
_. _.. ._..~" _. _ _ ~_...._~._
Se uence' y Y,rv . y .' OgE Start at 1 ORF Stop at 2329
TCAATTGACTGGCCACCCTGTCCTCACGTGTCAA
TGGCGTCCGCCTCAACCTCAGCCTGCTGCAGACAGAGCCCTCT
TCACCATCTGGGATGGGCCACAGCAAACAGCACCACGGCTCGGCGTCTTCACCCGGAG
ATCCACTCACCAAATGCCCTCCTCCCACCATCCTC
193



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
C
,~,.", T .,.-.l,nnT T nmrnmra ra r am
ATCCAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCT
G
C
.._ ....,r ."r T "" ~",.,rt,nnrmrrrn~mr_r-
_armarmmc'C''TC'AGCGAGAAGCAATATGATGAGTTTGAGATTT
P.ACCAAGGCCATGTACAGCTGCAGTGAAGGCTACCACCTCCAGGCAGGCGCTGAGGCCACTGCAGAG
r
3TCTGGACACAGGCCTATGGAGCAACCGCAATGTCCCACCACAGTGTGTCCCTGTGACTTGTCCTGA
r
GTCAGTAGCATCAGCGTGGAGCATGGCCGATGGAGGCTTATCTTTGAGACACAGTATCAGTTCCAGG
C
CCAGCTGATGCTCATCTGTGACCCTGGCTACTACTATACTGGCCAAAGGGTCATCCGCTGTCAGGCC
CCCCCCAATGGCCACCGCATCGGAACACTGTCTGTCTACGGGGCAACAGCCATCTTCTCCTGCAATT
CTGAGCCCATTGTCAACGGACACATCAATGGGGAGAA
TGCAATGCTGGCTTCCGCCTGATCGGCATGTCTGTGC
c~AAGACCCCTTTCTGTGTGCCAATTACCTGTGGACAC
C
TGGCACATGGGACCGTCCCCGCCCCCAGT
OV4g, 273095147 ° SEQ ~ NO: 44 776 as MW at ~4741.1kI~
ANGIVRGAGYNVGQSVTFECLPGYQLTGHPVLTCQHGTNRNWDHSLPICCEVPCGGNI
FPSPYSSSQDCVWLITVPIGHGVRLNLSLLQTEPSGDFITIWDGPQQTAPRLGVFTR
S
194



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
IFAIAFSAYPLTKCPPPTILPNAEVWENEEFNIGDIVRYRC
L
PGFTLVGNEILTCKLGTYLQFEGPPPICEVHCPTNELLTDSTGVILSQSYPGSYPQFQTCSWLVRVE
DWISLTVEYFLSEKQYDEFEIFDGPSGQSPLLKALSGNYSAPLIVTSSSNSVYLRWSSDHAYNRKG
F
KIRYSAPYCSLPRAPLHGFILGQTSTQPGGSIHFGCNAGYRLVGHSMAICTRHPQGYHLWSEAIPLC
VSSISVEHGRWRLIFETQYQFQAQLMLICDPGYYYTGQRVIRCQANGKWSLGDSTPTCRIISCGELP
I
PPNGHRIGTLSWGATAIFSCNSGYTLVGSRVRECMANGLWSGSEVRCLAGHCGTPEPIVNGHINGE
SYRGSVWQCNAGFRLIGMSVRICQQDHHWSGKTPFCVPITCGHPGNPVNGLTQGNQFNLNDWKF
CNPGYMAEGAARSQCLASGQWSDMLPTCRIINCTDPGHQENSVRQVHASGPHRFSFGTTVSYRCNHG
F
YLLGTPVLSCQGDGTWDRPRPQCLCKVD
......,...~................ . ......... ............~~.. ."~"..
NOV4h, 317459653 SEQ m N0: 45 ...... 328 bp.....~.._~.....~..
.................._.................~.~ ......... ....._..
DNA Sequence ~ORF Start: at ~l ORF Stop: end of sequence
~.,.....~T
_ V -:-~.-«.
~.._..TGG
V ~CGTGG
~ACAA
AGC
YGGGG
iAAGCTTGAGTGCCCAGACCCAGAGCCCTTTGCCAATGGCATTGTGA
C
TCACCATCTGGGATGGGCCACAGCAAACAGCACCACGGCTCGGCGTCTTCACCCGGAG
C
TCCACTCACCAAATGCCCTCCTCCCACCATCCTC
C
CCAACGCCGAAGTCGTCACAGAGAATGAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCT
C
T~AGCCAGAGCTACCCTGGAAGCTATCCCCAGTTCCAGACCTGCTCTTGGCTGGTGAGAGTGGAGCC
TATGATGAGTTTGAGATTT
TCAGGACAGAGTCCTCTGCTGAAAGCCCTCAGTGGGAATTACTCAGCTCCCCTGATT
C
CCTCTCTGTCA
195



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
AACCAAGGCCGTGTACAGCTGCAGTGAAGGCTACCACCTCCAGGCAGGCGCTGAGGCCACTGCAGAG
T
GTCTGGACACAGGCCTATGGAGCAACCGCAATGTCCCACCACAGTGTGTCCCTGTGACTTGTCCTGA
T
GTCAGTAGCATCAGCGTGGAGCATGGCCGATGGAGGCTTATCTTTGAGACACAGTATCAGTTCCAGG
C
CCAGCTGATGCTCATCTGTGACCCTGGCTACTACTATACTGGCCAAAGGGTCATCCGCTGTCAGGCC
A
ATGGCAAATGGAGCCTCGGGGACTCTACGCCCACCTGCCGAATCATCTCCTGTGGAGAGCTCCCGAT
T
CCCCCCAATGGCCACCGCATCGGAACACTGTCTGTCTACGGGGCAACAGCCATCTTCTCCTGCAATT
C
CGGATACACACTGGTGGGCTCCAGGGTGCGTGAGTGCATGGCCAATGGGCTCTGGAGTGGCTCTGAA
G
TCCGCTGCCTTGCTGGACACTGTGGGACTCCTGAGCCCATTGTCAACGGACACATCAATGGGGAGAA
C
TACAGCTACCGGGGCAGTGTGGTGTACCAATGCAATGCTGGCTTCCGCCTGATCGGCATGTCTGTGC
G
CATCTGCCAGCAGGATCATCACTGGTCGGGCAAGACCCCTTTCTGTGTGCCAATTACCTGTGGACAC
C
CAGGCAACCCTGTCAACGGCCTCACTCAGGGTAACCAGTTTAACCTCAACGATGTGGTCAAGTTTGT
T
TGCAACCCTGGGTATATGGCTGAGGGGGCTGCTAGGTCCCAATGCCTGGCCAGCGGGCAATGGAGTG
A
CATGCTGCCCACCTGCAGAATCATCAACTGTACAGATCCTGGACACCAAGAAAATAGTGTTCGTCAG
G
TCCACGCCAGCGGCCCGCACAGGTTCAGCTTCGGCACCACTGTGTCTTACCGGTGCAACCACGGCTT
C
TACCTCCTGGGCACCCCAGTGCTCAGCTGCCAGGGAGATGGCACATGGGACCGTCCCCGCCCCCAGT
G
TCTCTGTAAGGTCGAC
NOV4h, 317459653 SEQ m NO: 46 776 as MW at ~4719.1kD
Protein,Sequence.._._.......
_......_...._....................._......_....__............._............
........ ......................... ......._._..........._._.._.._... .
_......_
KLECPDPEPFANGIVRGAGYNVGQSVTFECLPGYQLTGHPVLTCQHGTNRNWDHPLPKCEVPCGGNI
T
SSNGTVYSPGFPSPYSSSQDCWLIWPIGHGVRLNLSLLQTEPSGDFITIWDGPQQTAPRLGVFTR
S
MAKKTVQSSSNQVLLKFHRDAATGGIFAIAFSAYPLTKCPPPTILPNAEWTENEEFNIGDIVRYRC
L
PGFTLVGNEILTCKLGTYLQFEGPPPICEVHCPTNELLTDSTGVILSQSYPGSYPQFQTCSWLVRVE
P
DYNISLTVEYFLSEKQYDEFEIFDGPSGQSPLLKALSGNYSAPLIVTSSSNSVYLRWSSDHAYNRKG
F
KIRYSAPYCSLPRAPLHGFILGQTSTQPGGSIHFGCNAGYRLVGHSMAICTRHPQGYHLWSEAIPLC
Q
ALSCGLPEAPKNGMVFGKEYTVGTKAWSCSEGYHLQAGAEATAECLDTGLWSNRNVPPQCVPVTCP
D
VSSISVEHGRWRLIFETQYQFQAQLMLICDPGYYYTGQRVIRCQANGKWSLGDSTPTCRIISCGELP
I
PPNGHRIGTLSWGATAIFSCNSGYTLVGSRVRECMANGLWSGSEVRCLAGHCGTPEPIVNGHINGE
N
YSYRGSWYQCNAGFRLIGMSVRICQQDHHWSGKTPFCVPITCGHPGNPVNGLTQGNQFNLNDWKF
V
CNPGYMAEGAARSQCLASGQWSDMLPTCRIINCTDPGHQENSVRQVHASGPHRFSFGTTVSYRCNHG
F
YLLGTPVLSCQGDGTWDRPRPQCLCKVD
NOV4i, 317459612 SEQ m NO: 47 .2139 by
DNA Sequence ° ORF Start: at 1~ ORF Stop: end of sequence
196



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
CACCCCCTGCCCAAGTGTGAAGTCCCTTGTGGCGGGAACATCAC
ACTCCAGCTCCCAGGACTGTGTCT
TCACCGTGCCCATTGGCCATGGCGTCCGCCTCAACCTCAGCCTGCTGCAGACAGAGCCCTCT
TCCAACCAGGTCCTGCTCAAGTTCCACCGTGATGCAGCCA
TCCTC
TGAAGAATTCAATATAGGTGACATCGTACGCTACAGATGCCT
CCTGGCTTTACCTTAGTGGGGAATGAAATTCTGACCTGCAAACTTGGAACCTACCTGCAGTTTGAAG
r~
CCCCGATATGTGAAGTGCACTGTCCAACAAATGAGCTTCTGACAGACTCCACAGGCGTGATC
CAGAGCTACCCTGGAAGCTATCCCCAGTTCCAGACCTGCTCTTGGCTGGTGAGAGTGGAGCC
rAACATCTCCCTCACAGTGGAGTACTTCCTCAGCGAGAAGCAATATGATGAGTTTGAGATTT
3TCCATCAGGACAGAGTCCTCTGCTGAAAGCCCTCAGTGGGAATTACTCAGCTCCCCTGATT
TCGGAAGGGCTT
ACAGCTGCAGTGAAGGCTACCACCTCCAGGCAGGCGCT
TGGCCGATGGAGGCCTATCTTTGAGACAC
TCATCTCCTG
TGGGCTC
TTGTCAACGGACA
ACAGCTACCGGGGCAGTGTGGTGTACCAATGCAATGCTGGCTTCCGCCTGA
CGGCATGTCTGTGCGCATCTGCCAGCAGGATCATCACTGGTCGGGCAAGACCCCTTTCTGTGTGCCA
A
ACCTCCTGGGCACCCCAGTGCTCAGCTGCCAGGGAGATGGCACATGGGACC
197



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
G
TCCCCGCCCCCAGTGTCTCTGTAAGGTCGAC
NOV4i, 317459612 SEQ m NO: 48 '713 as ~MW at 77947.4kD
Protein Sequence ~ -_
KLECPDPEPFANGIVRGAGYNVGQSVTFECLPGYQLTGHPVLTCQHGTNRNWDHPLPKCEVPCGGNI
T
SSNGTVYSPGFPSPYSSSQDCVWLITVPIGHGVRLNLSLLQTEPSGDFITIWDGPQQTAPRLGVFTR
S
MAKKTVQSSSNQVLLKFHRDAATGGIFAIAFSAYPLTKCPPPTILPNAEWTENEEFNIGDTVRYRC
L
PGFTLVGNEILTCKLGTYLQFEGPPPICEVHCPTNELLTDSTGVILSQSYPGSYPQFQTCSWLVRVE
P
DYNISLTVEYFLSEKQYDEFEIFDGPSGQSPLLKALSGNYSAPLIVTSSSNSVYLRWSSDHAYNRKG
F
KIRYSALSCGLPEAPKNGMVFGKEYTVGTKAMYSCSEGYHLQAGAEATAECLDTGLWSNRNVPPQCV
P
VTCPDVSSISVEHGRWRPIFETQYQFQAQLMLICDPGYYYTGQRVIRCQANGKWSLGDSTPTCRIIS
C
GELPIPPNGHRIGTLSVYGATAIFSCNSGYTLVGSRVRECMANGLWSGSEVRCLAGHCGTPEPIVNG
H
INGENYSYRGSVVYQCNAGFRLIGMSVRICQQDHHWSGKTPFCVPITCGHPGNPVNGLTQGNQFNLN
D
VVKFVCNPGYMAEGAARSQCLASGQWSDMLPTCRIINCTDPGHQENSVRQVHASGPHRFSFGTTVSY
R
CNHGFYLLGTPVLSCQGDGTWDRPRPQCLCKVD
NOV4j, 317286331 SEQ m NO: 49 2607 by
DNA Sequence ORF Start: at 1 ORF Stop: end of sequence
a
AAGCTTTCATGTTTTGATCCTGGTTCCATCAAGAACGGCACACGGGTGGGGTCCGACCTGAAGCTGG
G
CTCCTCCGTCACCTACTACTGCCACGGGGGCTACGAAGTTGAGGGCACCTCGACCCTGAGCTGCATC
C
TGGGGCCTGATGGGAAGCCCGTGTGGAACAATCCCCGGCCAGTCTGCACAGCCCCCTGTGGGGGACA
G
TATGTGGGTTCGGACGGAGTGGTCTTGTCCCCCAACTACCCCCAGAACTACACCAGTGGACAGATCT
G
CTTGTATTTTGTTACTGTGCCCAAGGACTATGTGGTGTTTGGCCAGTTCGCCTTCTTTCACACGGCC
C
TCAACGACGTGGTGGAGGTTCACGACGGCCACAGCCAGCACTCGCGGCTCCTCAGCTCCCTCTCGGG
C
TCCCATACAGGAGAATCACTGCCCTTGGCCACCTCCAATCAAGTTCTCATTAAGTTCAGCGCCAAAG
G
CCTCGCACCAGCCAGAGGCTTCCACTTTGTCTACCAAGCGGTTCCTCGAACCAGCGCCACGCAGTGC
A
GCTCTGTGCCGGAACCCCGCTATGGCAAGAGGCTGGGCAGTGACTTCTCGGTGGGGGCCATCGTCCG
C
TTCGAATGCAACTCCGGCTATGCCCTGCAGGGGTCGCCAGAGATCGAGTGCCTCCCTGTGCCTGGGG
C
CTTGGCCCAATGGAATGTCTCAGCGCCCACGTGTGTGGTGCCGTGTGGAGGCAACCTCACAGAGCGC
A
GGGGCACCATCCTGTCCCCTGGCTTCCCAGAGCCGTACCTCAACAGCCTCAACTGTGTGTGGAAGAT
C
GTGGTCCCCGAAGGCGCTGGCATCCAGATCCAAGTTGTCAGTTTTGTGACAGAGCAGAACTGGGACT
C
GCTGGAAGTATTTGATGGTGCAGATAACACTGTAACCATGCTGGGGAGTTTCTCAGGAACAACCGTG
C
CTGCCCTTCTGAACAGCACCTCCAACCAGCTCTACCTTCATTTCTACTCAGATATCAGCGTATCTGC
A
GCTGGCTTCCACTTGGAGTACAAAACGGTGGGCCTGAGCAGTTGTCCGGAACCTGCTGTGCCCAGTA
198



CA 02488547 2004-12-02
WO 03/102155 PCT/US03/17430
A
CGGGGTGAAGACTGGCGAGCGCTACTTGGTGAATGATGTGGTGTCTTTCCAGTGTGAGCCGGGATAT
TGGACTGCTCCTGGAAAATAGCACTGCCCGTGGGCTTTGGAGCTCAC
ATCAGGCC
CAGAGCCCTTTGCCAATGGCATTGTGAGGGGAGCTGGCTACAA
TCAGTGACCTTCGAGTGCCTCCCGGGGTATCAATTGACTGGCCACCCTGTCCTCACGT
G
TCAACATGGCACCAACCGGAACTGGGACCACCCCCTGCCCAAGTGTGAAGTCCCTTGTGGCGGGAAC
CTCAACCTCAGCCTGCTGCAGACAGAGC
TTTCATCACCATCTGGGATGGGCCACAGCAAACAGCACCACGGCTCGGCGTCTTCACC
CGTGATGC
A
GCCACAGGGGGGATCTTCGCCATAGCTTTCTCCGCTTATCCACTCACCAAATGCCCTCCTCCCACCA
TATGTGAAGTGCACTGTCCAACAAATGAGCTTCTGACAGACTCCACAGGCG
CAGACCTGCTCTTGGCTGGTGAGAGTG
CGACTATAACATCTCCCTCACAGTGGAGTACTTCCTCAGCGAGAAGCAATATGATGAGTTTGA
TCAGGACAGAGTCCTCTGCTGAAAGCCCTCAGTGGGAATTACTCAGCTCCCC
TTGTCACCAGCTCAAGCAACTCTGTGTACCTGCGTTGGTCATCTGATCACGCCTACAATCGGAAG
V4j, 31726331 y ~Q m NO: 50 X69 as ~MW at 94553.SkD
PGSIKNGTRVGSDLKLGSSVTYYCHGGYEVEGTSTLSCILGPDGKPVWNNPRPVCTAPCGG
WLSPNYPQNYTSGQICLYFVWPKDYWFGQFAFFHTALNDWEVHDGHSQHSRLLSSLS
LPLATSNQVLIKFSAKGLAPARGFHFVYQAVPRTSATQCSSVPEPRYGKRLGSDFSVGAIV
PVPGALAQWNVSAPTCWPCGGNLTERRGTILSPGFPEPYLNSLNCWK
IQIQWSFVTEQNWDSLEVFDGADNTVTMLGSFSGTTVPALLNSTSNQLYLHFYSDISVS
VPSNGVKTGERYLVNDWSFQCEPGYALQGHAHISCMPGTVRRWNYPP
199




DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 9
CONTENANT LES PAGES 1 A 199
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 9
CONTAINING PAGES 1 TO 199
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE: