CA2446437A1 - Novel antibodies that bind to antigenic polypeptides, nucleic acids encoding the antigens, and methods of use - Google Patents

Abstract

Disclosed herein are nucleic acid sequences that encode polypeptides. Also disclosed are antibodies, which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. 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, polypeptides, or antibodies, or fragments thereof.

Description

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NOVEL ANTIBODIES THAT BIND TO ANTIGENIC POLYPEPTIDES, NUCLEIC
ACIDS ENCODING THE ANTIGENS, AND METHODS OF USE
FIELD OF THE INVENTION
The present invention relates to novel antibodies that bind immunospecifically to antigenic polypeptides, wherein the polypeptides have characteristic properties related to biochemical or physiological responses in a cell, a tissue, an organ or an organism. The novel polypeptides are gene products of novel genes, or are specified biologically active fragments or derivatives thereof. Methods of use of the antibodies encompass procedures for diagnostic and prognostic assay of the polypeptides, as well as methods of treating diverse pathological conditions.
BACKGROUND 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 patlr6lagies the dysregulation is manifested as elevated or excessive synthesis and secretion of protein effectors. In a clinical setting a subject may be suspected of suffering from a condition brought on by elevated or excessive levels of a protein effector of interest.
Antibodies are multichain proteins that bind specifically to a given antigen, arid 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.
S 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.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOV l, NOV2, NOV3, etc., nucleic acids and polypeptides.
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.
In one aspect, the invention provides an isolated palypeptide comprisi.»b a :::~t~,:.re form of a NOVX amino acid. The polypeptide can be, for example, a NOVX amino acid sequence or a variant of a NOVX amino acid sequence, 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. The invention also includes fragments of any of NOVX polypeptides. In another aspect, the invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof.
Also included in the invention is a NOVX polypeptide that is a naturally occurring variant of a NOVX sequence. In one embodiment, the variant includes an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a NOVX nucleic acid sequence. In another embodiment, the NOVX polypeptide is a ' variant polypeptide described therein, wherein any amino acid specified in the chosen sequence is changed to provide a conservative substitution.
In another aspect, invention provides a method for determining the presence or amount of the NOVX polypeptide in a sample by providing a sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the NOVX polypeptide, thereby determining the presence or amount of the NOVX polypeptide in the sample.
In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX
polypeptide in a mammalian subject by measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and comparing the amount of the polypeptide in the sample of the first step 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. 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 aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier. The therapeutic can be, e.g., a NOVX
nucleic acid, a ' NOVX polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylaetically-effective amount of this pharmaceutical composition.
In still another aspect, the invention provides the use of a therapeutic in the ar!anufacture of a medicament for treating a syndrome associated :vi~h a h~amar. dise~ ~~:
is associated with a NOVX polypeptide.
In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample expressing the NOVX polypeptide with antibody that binds the NOVX polypeptide in an amount sufficient to modulate the activity of the polypeptide.
The invention also includes an isolated nucleic acid that encodes a NOVX
polypeptide, or a fragment, homolog, analog or derivative thereof. In a preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.. In another embodiment, the nucleic acid encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant. In another embodiment, the nucleic acid molecule differs by a single nucleotide from a NOVX nucleic acid sequence. In one embodiment, the NOVX 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 46, or a complement of the nucleotide sequence. In one embodiment, the invention provides a nucleic acid molecule wherein the nucleic acid includes the nucleotide sequence of a naturally occurring allelic nucleic acid variant.
Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
The invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
In yet another aspect, the invention provides for a method for determining the presence or amount of a nucleic acid molecule in a sample by contacting a sample with a probe that binds a NOVX nucleic acid and determining the amount of the probe that is bound to the NOVX nucleic acid. For example the NOVX nucleic may be a marker for cell or tissue type such as a cell or tissue type that is cancerous.
In yet a further aspect, the invention provides a method for determining the presence of or predisposition to a disease associated with altered levels of a nucleic acid molecule in a first mammalian subject, 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 polypPptide. The NOVX antibody may be monoclonal, humanized, or a _ful_ly human antibody. Preferably, the antibody has a dissociatdor: crustant fo: ~~:e ~u.ding 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.
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 polypeptides, antibodies, and other related compunds. 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 1 provides a summary of the NOVX nucleic acids and their encoded polypeptides.
TABLE 1. NOVX Polynucleotide and Polypeptide Sequences and Corresponding SEQ ID Numbers SEQ
ID

Internal Identificationn ep Homology O e Assignment (n (po p c tid leic acid CG100051-02 RECEPTOR like, homo sa iens 2a 3 4 fibronectin-malate dehydrogenase like, homo CG100104-01 sa iens 2b 5 6 fibronectin-malate . dehydrogenase like, homo 198362674 sa iens 2c 7 8 fibronectin-malate dehydrogenase like, homo 198362686 sa iens MOLECULE

4a 11 12 Leucine Rich Repeat Membrane CG100619-01 ' Protein like, homo sa lens 4b 13 14 Leucine Rich Repeat Membrane 210168777 Protein like, homo sa lens 15 16 GTP:AMP phosphotransferase CG56785-01 mitochondrial like, homo sa lens 6a 17 18 Thrombospondin like, homo CG56914-01 sa lens 6b CG56914-02 19 20 Fibuiin tike, homo sa lens 7 CG57242-01 21 22 KIAA0900 like, homo sa lens 8a 23 24 Complement Decay-Accelerating Factor like, homo CG57279-02 sa lens 8b 25 26 Complement Decay-Accelerating Factor like, homo CG57279-04 sa lens 8c 27 28 . Complement Decay-Accelerating Factor like, homo CG57279-05 sa lens 8d 29 30 Complement Decay-Accelerating Factor like, homo 175070639 sa lens like, CG94630-01 homo sa lens 10a CG94831-01 33 34 Tetras an-2 like, homo sa lens IOb CG94831-02 35 36 Tetras an-2 like, homo sa lens 11 37 38 CUB domain containing membrane protein like, homo CG94892-01 sa lens 12a 39 40 Collagen alpha 2 (VIII) chain CG95227-01 like, homo sa lens 12b 41 42 Collagen alpha 2 (VIN) chain CG95227-02 tike, homo sa lens 13a CG96384-01 43 44 Plasma Membrane Protein 13b CG96384-02 45 46 Plasma Membrane Protein 13c 209749131 47 48 Plasma Membrane Protein 13d 209749030 49 50 Plasma Membrane Protein 14 ( 51 52 ~ SodiumlHydrogen Exchanger CG96432-01 like, ( Fomo sa i2i ~s 15a CG96545-02 53 54 EPHRIN-A5 PRECURSOR

ISb CG96545-03 55 56 EPHRiN-A5 PRECURSOR

CG97101-01 RELATED like, homo sa lens 17 59 60 ATP-Binding Cassette CG97168-01 trans orter A like, homo sa lens 18a 61 62 MAGE-domain Containing like, CG97420-01 homo sa lens 18b 63 64 MADE-domain Containing like, CG97420-02 homo sa lens 19a 65 66 collagen and scavenger receptor CG97430-01 domain like, homo sa lens 19b 67 68 collagen and scavenger receptor CG97430-02 domain like, homo sa lens 20a 69 70 CUB domain-containing like, CG97440-01 homo sa lens 20b 71 72 CUB domain-containing tike, 199652779 homo sa lens 21 CG97451-01 73 74 GI cine-rich membrane rotein like, homo sa iens 22a CG97852-01 75 76 atectin 9 tike, homo sa iens 22b CG97852-03 77 78 alectin 9 like, homo sa iens 23a 79 80 T Cetl Surface Glycoprotein like, homo sa iens 23b 81 82 T Cell Surface Glycoprotein like, homo sa iens 24 83 84 1110002C08RtK PROTEIN
CG99608-01 tike, homo sa iens 25 85 86 EPITHELIAL V LIKE, PRECURSOR

26a CG99732-02 87 88 MACROPHAGE LECTIN 2 26b CG99732-03 89 90 MACROPHAGE LECTIN 2 27 CG99767-01 9~ 92 . a I membrane rotein Table 1 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 1 will be useful in therapeutic and diagnostic applications implicated in, for example, pathologies and disorders associated with the known protein families identified in column 5 of Table 1.
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 1, 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 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 1.
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.
I 5 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 research tools. 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 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 ID
NO: 2n, wherein n is an integer between 1 and 46; (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 46, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 1 S% 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 ID NO: 2n, wherein n is an integer between 1 and 46; (d) a variant of the S amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 46 wherein 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; 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 46; (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID
NO: 2n, wherein n is an integer between 1 and 46 wherein any amino acid in the mature 1 S form of 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 of the mature form are so changed; (c) the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between l and 46; (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 46, 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;
(e) a nucleic acid fragment encoding at least a portion of a nolypeptide comprising the amino acid sequence selected from the group consisting of S>~:Q _Tn NC)' Vin, Wherein n ;~ an integer between 1 and 46 or any variant of said polypeptide wherein any amino acid of the 2S 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.
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.46; (b) a nucleotide sequence 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 46 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than I S% 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 46; 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-1, wherein n is an integer between 1 and 46 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 mRNA's) 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 occurnng polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein ineludc-s~, 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, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host 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 polypeptide or protein that has residues I 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 I 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 "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 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- 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 utilized herein, is one, which 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, I kb9 0.5 kb or 0.1 kb of nucleotide sequences which naturally fl_ auk the nucleic acid molecule in genomic DNA of the cPl1/tiss»e from which the nT~ele~e 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 when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1-46, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
Using all or a portion of the nucleic acid sequence of SEQ ID N0:2n-1, wherein n is an integer between 1-46, 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 2°d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; 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 and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to 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, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomie or eDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, SO 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 1-46, 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 SEQ ID
N0:2n-l, wherein n is an integer between 1-46, or a portion nftl~is _n_ncleot~d~Pq"PnCe (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-l, wherein n is an integer between 1-46, is one that is su~ciently complementary to the nucleotide sequence of SEQ
ID NO:2n-l, wherein n is an integer between 1-46, that it can hydrogen bond with little or no mismatches to the nucleotide sequence of SEQ ID N0:2n-l, wherein n is an integer between 1-46, 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 S without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial.
substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect 1 S to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
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 S' direction of the disclosed sequence. Any disclosed NOVA nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective 2S NOVX polypeptide, and requires that the corresponding full-length cDNA
extend in the 3' direction of the disclosed sequence.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 9S%
identity (with a preferred identity of 80-9S%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. 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 encode 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-I, wherein n is an integer between 1-46, as well as a polypeptide possessing NOVX
biological activity. Various biological activities of the NOVX proteins are described below.
A NOVX polvpeptide 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 OR,F
is uninterrupted by a stop colon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" colon and terminates with one of the three "stop"
colons, namely, TAA, TAG, or TGA. For the purposes of this invention, an OR.F
may be any part of a coding sequence, with or without a start colon, a stop colon, 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 o$en 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 andlor cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX

homologues from other vertebrates. The p~obe/primer 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
ID N0:2n-l, wherein n is an integer between 1-46; or an anti-sense strand nucleotide sequence of SEQ >D N0:2n-1, wherein n is an integer between 1-46; or of a naturally occurring mutant of SEQ ID N0:2n-1, wherein n is an integer between 1-46.
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 further comprises a label group attached thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-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-1, wherein n is an integer between 1-46, that encodes a polypeptide having a NOVX
biological activity (the biological activities of the NOVX proteins are described below), ea~pressing the encoded portion of NOVX protein (e.g., by recombinant expression ~ vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ TD N0:2n-1, wherein n is an integer between 1-46, due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between 1-46. 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 ID N0:2n, wherein n is an integer between 1-46.

In addition to the human NOVX nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between 1-46, 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 S 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 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 any one of the human SEQ ID
NO:2n-1, wherein n is an integer between 1-46, 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 sennPncP of SEQ _TD NO:2n-1, wherein n is an integer between 1-46. 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 60% homologous to each other typically remain hybridized to each other.
Fiomologs (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 I 0 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, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 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 B10LOGY, john Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, 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, (1.52% WSA, and 500 mg/ml denal,~rerl salmon sne,_m 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 any one of the sequences of SEQ ID N0:2n-l, wherein n is an integer between 1-46, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between I-46, 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 SS °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
S 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 ID N0:2n-l, wherein n is an integer between I-46, 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 3S% formamide, SX SSC, SO mM Tris-HCI (pH 7.S), S mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.2°/ BSA, I00 mglml denatured salmon sperm DNA, 10%
(wt/volt) dextran sulfate at 40 °C, followed by one or more washes in 2X SSC, 2S mM
Tris-HCl (pH 7.4), S mM EDTA, and 0.1% SDS at SO °C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species 1 S hybridizations). See, e.g., Ausubel, et al. (eds.),1993, CURRENT PROTOCOLS
IN
MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER
AND
ElIPRESSIQN, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981.
Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations In addition to naturally-occurring 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 sequence~~~of SEQ ID N0:2r_-J , WhPrP;" ~ is ~
integer between 1-46, thereby leading to changes in the amino acid sequences of the 2S encoded NOVX proteins, without altering the functional ability of said NOVX
proteins.
For example, nucleotide substitutions leading to amino acid substitutions at "non-essential"
amino acid residues can be made in the sequence of SEQ ID NO:2n, wherein n is an integer between 1-46. 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 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 any one of SEQ ID NO:2n-I, wherein n is an integer between 1-46, yet retain biological activity. In one embodiment, the isolated S nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID N0:2n, wherein n is an integer between 1-46.
Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID
N0:2n, wherein n is an integer between 1-46; more preferably at least about 70%
homologous to SEQ ID N0:2n, wherein n is an integer between 1-46; still more preferably at least about 80% homologous to SEQ ID N0:2n, wherein n is an integer between 1-46;
even more preferably at least about 90% homologous to SEQ ID NO:Zn, wherein n is an integer between 1-46; and most preferably at least about 95% homologous to SEQ
ID
N0:2n, wherein n is an integer between 1-46.
An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID N0:2n, wherein n is an integer between 1-46, 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 1-46, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into any of SEQ ID NO:2n-1, wherein n is an integer between 1-46, 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 a=mino acid substitution" i~
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 any one of SEQ ID N0:2n-I, wherein n is an integer between 1-46, 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, NEQK, 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 be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, 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).
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 ID N0:2n-1, wherein n is an integer between 1-46, 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, S0, 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 ID N0:2n, wherein n is an integer between 1-46, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID N0:2n-1, wherein n is an integer between 1-46, 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 mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modif ed nucleotides designed to increase the biological stability of the molecules or to increase the physicaa 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: S-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, l-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, S-methylaminomethyluracil, S-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3 N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. 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 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 de1_ivered to cells using the vectors described herein. To ach;PVe sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pot 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., any one of SEQ ID
NO:Zn-l, wherein n is an integer between 1-46). For example, a derivative of a Tetrahymena 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 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 rev?gin 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.
Anticaneer 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. Bioorg Med 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.
S 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 antigens 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., Sl nucleases (See, Hyrup, et al., I996.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 1 S 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
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 teases, and orientation (see, Hyrup, et al., 1996. supra). The syntfi-esis of PNA-DNA ch?t?-!Pras ran be performed as describPri in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For 2S example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., S'-(4-methoxytrityl)amino-S'-deoxy-thymidine phosphoramidite, cari be used between the PNA and the S' 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 S' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra.
Alternatively, chimeric molecules can be synthesized with a S' DNA segment and a 3' PNA
segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lets. S: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acad. Sci. U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
W088/09810) 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 S (see, e.g., Krol, et a1.,1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. S: 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 ID N0:2n, wherein n is an integer between 1-46. 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 1-46, while 1 S 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 amino acids, and fi.wther include the possibility of inserting an additional residue or residues between taro 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-2S 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. In 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%
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 e~mYr_cing 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 N0:2n, wherein n is an integer, between 1-46) that include fewer amino acids than the full-length NOVX
proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active port' ions 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.

In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID
N0:2n, wherein n is an integer between 1-46. In other embodiments, the NOVX
protein is substantially homologous to SEQ ID NO:2n, wherein n is an integer between 1-46, and retains the functional activity of the protein of SEQ ID N0:2n, wherein n is an integer between 1-46, 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 ID NO:2n, wherein n is an integer between 1-46, and retains~the functional activity of the NOVX proteins of SEQ ID
N0:2n, wherein n is an integer between 1-46.
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 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 ]der~t,_ty between _ __.two sequences. The homology may be determined using computer r_rog_ramc 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 NO:2n-l, wherein n is an integer between 1-46.
The team "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 g0 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 N0:2n, wherein n is an integer between 1-46, 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 a NOVX
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 iwo biologically-active portions of a NOVX prote~_ pn~yet ~nnther 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-immunoglobuIin 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 enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic liga+.lon. In another embodiment, the fusion gene car+ be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, FC..R
_amplification ~f 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 occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurnng 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 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 t:r .produce libraries of potential NOVX ~~a.~iants from a degenerate oligonucleotide sequer_ce_ ~'hP~nica~ synthesis of a degenerate gene sequence can bP
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. A~nu. Rev.
Bioehem. 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 S~
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 replicable 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 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.
NOVX Antibodies 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(a6~2 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 IgG~, 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 immunospecificalIy 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 1-46, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the i~~1_i_ 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 indicate which regions of a NOVX polypeptide are particularly hydrophilic and, therefore, encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophiiicity 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:

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 polyppeptide 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 pM, preferably <_ 100 nM, more preferably <_ 10 nM, and most preferably <_ 100 pM to about 1 pM, as S measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.
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 1 S 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 immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed imununogenic prote3:~. Furt~hernore, ~e protei:~ 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 parvum, 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 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 Kohler and Milstein, Nature, 256:495. (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically i~: m~arized with an immunizing agent to elicit lymphocytes that produce or are capable of rrouuci~~g 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-def cient 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 marine 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 (I~ozbor, 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 PoIIard, Anal, Biochem.,107:220 (1980). It is an objective, especiahy ~inportant r"
therape"t;c 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 irnmunoglobulin 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; Morrison, Nature 368, (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 im_mt~~e response by the h»xnan aga~net the a~m?l~~StPred immunoglobulin. Humanized forms of antbodies arP crdmeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'~ 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 a1.,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 triorna technique;
the human B-cell hybridoma technique (see Kozbor, 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 a1.,1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et 2O al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. L1SS, Inc., pp:
77-96).
In addition, human antibodies can also be produced using additional technictues, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
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)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-SI (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol.

(1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the S 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 immunoglobuli_n heavy chain is disclosed in T T.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 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 F~, fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~ab~2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab72 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 spP~ificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
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-(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 Iarge 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.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab'')a bispecific antibodies). Techniq~ae~ for generating bispecific antibodies from antibody fragments have been described in the literature. For example, 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 Flab'}a fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular 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')a 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 Ft. \yF,~) d;mPrS has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
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), FcyRII
(CD32) and FcyRIII (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 (tT.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: 2918-2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et a1. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement Iysis 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 WO 02/090504 PCT/US02/14342 .-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 zl2Bi, i3ih l3iln, 9oY, and lasRe.
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 W094111026.
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
S 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 Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, fox use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain, are utilized as pharmacologically-active compounds (see below).
An antibody specific fox a protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffmity chromatography or immunoprecipitation.
Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein.
Antibodies directed against the 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, Iuciferin, and aequorin, and examples of suitable radioactive material include lash 1311, 3sS 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 subj ect. 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. Tn the first instance, 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 occurnng ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which Iigand 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. Tn 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 obj ective. 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 Pxactice Of Pharmacy 19th ed. (Alfonso R. Gennaxo, 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 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-(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-(methylmethacrylate) 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 _ microcapsuIes. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),, or poly(vinylalcohol)), polylactides (U.S.
I O 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 I S over 100 days, certain hydrogels release proteins for shorter time periods.
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 20 polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or Flab>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, ~s'w~ll as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
Examples of 25 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 30 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 in vitro as well as in 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, irnmunoprecipitations, 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 Thory 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 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 episomai manunali~u-~ vectois).
~th er ~~ctns (e.g., non-episomal mammalian vectors) are integrated mto 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).
I O 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 IN 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, 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 nrmtPj~S, Pty':).
The recombinant expression vectors of the inver_tion ran bP designed fox 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 carried out in Escherichia eoli 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. eoli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11 d (Studier et al., GENE
E3~PRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CaIif.
(1990) I S 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
IN
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: 2111-2118). Such alteration of nucleic acid-sequences of the invention can be carried out by standard DN A s; nthesis techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces 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).
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
(Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable 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. Immunol.
43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO
J. 8: 729-733) and immunoglobulins (Banerji, et a1.,1983. Cell 33: 729-740;
Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-speci$c promoters (e.g., milk whey promoter; IT.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.~ , the marine hox promoters (I~essel and Grass, 1990. Science 249:
374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. P'enes nev 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 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, VoI. I (I ) 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.
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 trar~fecting 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 fiu-ther 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 I S 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 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 exarryics 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 ID
N0:2n-1, wherein n is an integer between 1-46, 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 and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding 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, 0.g., ~:nctioraily di~i-apt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of any one of SEQ ID N0:2n-I, wherein n is an integer between 1-46), 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-1, wherein n is an integer between I-46, 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 carried 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 S'- 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 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. O~ain.
Biotechnol. 2:
823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO
92/0968;
and WO 93/04169. w --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 a1.,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, orie 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 dl., 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 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), transdermaI
(i. e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, 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 ethylenediaminetetraacetie 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 EL'~ (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 carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, 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, pcly,~.lecl~ols auc:: ~a :nanit3l, 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 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 earner. 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 earner 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.
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 barrier 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 thrnn~ the base ef nasal sprays or suppositories. For transdermaI 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 carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subj ect 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.
USA 91:
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 pl:a..rrnaceutical preparation can include one or more Jells that produce the oenP dP1_1vP_r~,' ~- --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 insu~cient 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.
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 thereoL 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.
Anticancer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 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 S art, for example in: DeWitt, et a1.,1993. Proc. Natl. Acad. Sci. U.S.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. Ange~rv.
Chem. Int. Ed.
Engl. 33: 2059; Carell, et al., 1994. Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: SSS-SS6), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, 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. Science 249: 386-390; Devlin, 1990.
1S Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.SA.
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 enzyniafic label Sorb +.hat binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in 2S a complex. For example, test compounds can be labeled with Iash 3sS, ~4C, 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 WO 02/090504 PCT/US02/14342 .. ..,..-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 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 Ca2+, 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., Iuciferase), 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 I O 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.
In still another erribodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX cari 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 catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the retl_-free assay comprises contacting tie 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-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 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'ue uissociated from the matrix, and the levee of NC1VX r~rr~tei_rl h,'_nding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the 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 streptavidm-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.
S 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
mRIVA
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. T'he 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 a1.,1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogene 8: 1693-1696; and Brent WO 94/10300), to :dPr'ta~ -tier 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 WO 02/090504 PCT/US02/14342- _ .....w 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.
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 SEA 1 D N0:2n-l, wherein n is an integer between 1-46, 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., 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of 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 in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical 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?~e idPnt;fPd 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 WO 02/090504 PCT/US02/14342 _ .~.".
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 McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Fiopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a 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 qne or more restriction enzymes, and probed on a Southern blot to yield unique band.
for identification. The sequences of the invention are useful as additional DNA markers for RFLP ("restriction fragment length polyrnorphisms," 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 identif cation 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.
I 0 Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel ofperhaps 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 ~ is an integer between 1-46, 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 biol~gi~~1 sample (~.~ , blood, serum, cells, tissue) to thereby determine whether a~n 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 WO 02/090504 PCT/US02/14342 _ .. ....".
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").
Pharnacogenomics 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.) .
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., mIZNA, 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 N0:2n-1, wherein n is an integer between 1-46, ox a r onion thei:eof; 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')2) 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 i~ situ hybridizations. In vitro techniques for detection of NOVX
protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of 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 ~f 1':CtVX 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 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 activityl.
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 Ievel 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, (viiz~ 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.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polyrnerase 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. Acad. 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 ampli~catiowstep z~ co~?junction with any of the techniques used for detectir_g mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. PYac. 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. BioTechnolo~ 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 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. Sci. USA 74: 560 or Sanger, 1977. Proc.
Natl. Acad.
Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995. Biotechniques 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. Biotechnol. 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 RNA/DNA 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, RNAfDNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Sl nuclease to enzyrnatically 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 by size on denaturing polyacrylamide gels to determine the site of mutation.
See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 8S: 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. Carcinogenesis 1S:
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). 'lie duplex is treated ~,~~ah a DNA mismatch repair enzyme, and uhe 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. Anal..
Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX
nucleic acids will be denatwed 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: S.
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 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. Chem. 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. Acid. 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, ailtie specif a a,npli~cation-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 polymerise 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. Acid. Sei. 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.
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 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.) 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 compn~.~nd 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 pharmacogenornics of the individual perrriits 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. Physiol., 23: 983-985;
Linder, 1997.
Clin. Chem., 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 polymozphisms. 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.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duxation 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 metabolizer (EM) and poor metabolizer (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 CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a mctawlite is the active _~ __ r_ tthPra,~utac mrripty~ pM
show ne ther%~pe~at_ic 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, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subj ect 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, 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, fox example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes i-ml I»ated ~ 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 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 cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoaguladon, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.
These methods of treatment will be discussed more fully, below Disease 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; (iz~ 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. Science 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.
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 m>ZNAs (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 WO 02/090504 ._ ._, ._ _ PCT/US02/14342 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 of such inhibitory agents include antisense NOVX nucleic acid molecules and anti NOVX
antibodies. These modulatory methods can be performed i~ 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-regi.~lates 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 and/or in which increased NOVX activity has 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 may 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 including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, 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.
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 need thereof. By way of non-limiting example, the compositioii~ of the invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias.
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, WO 02/090504 .. .._.- ... _ .-.-- PCT/US02/14342 which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
EXAMPLES
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 1 A.
Table 1A.
NOVl Sequence Analysis SEQ ID NO: I I 127 by NOVla, CATATCACCAGTGGCCATCTGAGGTGTTTCCCTGGCTCTGAAGGGGTAGGCACGATGG

DNA Sequence CCAAGGCTCTTTGCGTGCAGAAGAGCTTTCCATCCAGGTGTCATGCAGAATTATGGGA

CTAAGTTTGGCCGGCAAGGACCAAGTTGAAACAGCCTTGAAAGCTAGCTTTGAAACTT

GCAGCTATGGCTGGGTTGGAGATGGATTCGTGGTCATCTCTAGGATTAGCCCAAACCC

CAAGTGTGGGAAAAATGGGGTGGGTGTCCTGATTTGGAAGGTTCCAGTGAGCCGACAG

TTTGCAGCCTATTGTTACAACTCATCTGATACTTGGACTAACTCGTGCATTCCAGAAA

TTATCACCACCAAAGATCCCATATTCAACACTCAAACTGCAACACAAACAACAGAATT

TATTGTCAGTGACAGTACCTACTCGGTGGCATCCCCTTACTCTACAATACCTGCCCCT

ACTACTACTCCTCCTGCTCCAGCTTCCACTTCTATTCCACGGAGAAAAAAATTGATTT

GTGTCACAGAAGTTTTTATGGAAACTAGCACCATGTCTACAGAAACTGAACCATTTGT

TGAAAATAAAGCAGCATTCAAGAATGAAGCTGCTGGGTTTGGAGGTGTCCCCACGGCT

CTGCTAGTGCTTGCTCTCCTCTTCTTTGGTGCTGCAGCTGGTCTTGGATTTTGCTATG

TCAAAAGGTATGTGAAGGCCTTCCCTTTTACAAACAAGAATCAGCAGAAGGAAATGAT

CGAAACCAAAGTAGTAAAGGAGGAGAAGGCCAATGATAGCAACCCTAATGAGGAATCA

AAGAAAACTGATAAAAACCCAGAAGAGTCCAAGAGTCCAAGCAAAACTACCGTGCGAT

GCCTGGAAGCTGAAGTTTAGATGAGACAGAAATGAGGAGACACACCTGAGGCTGGTTT

CTTTCATGCTCCTTACCCTGCCCCAGCTGGGGAAATTCAAAAGGGCCAAAGAACCAAA

GAAGGAAAGTCCACCCTTGGTTCCTAACTGGGATTCAGCTCAGGGACTGCCATTTGGA

CTATTGGGAGTTGCACCAAAGGAGA

ORF Start: ATG at ORF Stop: TAG at 946 SEQ ID NO: 2 297 as MW at.32454.8kD

NOVla, MARCFSLVLLLTSIWTTRLLVQGSLRAEELSIQVSCRIMGLSLAGKDQVETALKASFE

Protein SequenceEIITTI~PIFNTQTATQTTEFIVSDSTYSVASPYSTIPAPTTTPPAPASTSIPRRKKL

ICVTEVFMETSTMSTETEPFVENKAAFKNEAAGFGGVPTALLVLALLFFGAAAGLGFC

YVKRYVKAFPFTNKNQQKEMIETKVVKEEKANDSNPNEESKKTDKNPEESKSPSKTTV

RCLEAEV

Further analysis of the NOV 1 a protein yielded the following properties shown in Table 1 B.

Table 1B. Protein Sequence Properties NOVla PSort 0.4600 probability located in plasma membrane; 0.1000 probability located in analysis: endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in outside SignalP Cleavage site between residues 27 and 28 analysis:
A search of the NOV 1 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.
Table 1C. Geneseq Results for NOVla NOVla Identities/

Geneseq Protein/Organism/Length (PatentResidues/SimilaritiesExpect #, for IdentifierDate] Match the Matched Value ResiduesRegion AAB80247Human PR0263 protein - Homo I ..297 297/322 (92%)e-167 Sapiens, 322 aa. [WO200104311-Al, 1..322 297/322 (92%) AAB88391Human membrane or secretory 1..297 297/322 (92%)e-167 protein clone PSEC0135 - Homo sapiens,1..322 297/322 (92%) 322 aa.

[EP1067182-A2, 10-JAN-2001]

AAB87528Human PRO263 - Homo Sapiens,1..297 297/322 (92%)e-167 322 aa.

[WO200116318-A2, 08-MAR-2001]1..322 297/322 (92%) AAY87287Human signal peptide containing1..297 297/322 (92%)( e-167 protein HSPP-64 SEQ ID NO:64 - Homo 1..322 297/322 (92%) Sapiens, 322 aa. [wO200000610-A2, 2000]

AAY13379Amino acid sequence of protein1..297 297/322 (92%)e-167 - Homo Sapiens, 322 aa. [W09914328-1..322 297/322 (92%) A2, 25-MAR-1999]

In a BLAST search of public sequence datbases, the NOV 1 a protein was found to have homology to the proteins shown in the BLASTP data in Table 1 D.

Table 1D. Public BLASTP Results for NOVla Identities/

Protein Similarities NOVla Residues/ Expect AccessionProtein/Organism/Length Match Residuesfor the Value Number Matched Portion Q9UNF4 HYALURONIC ACID RECEPTOR 1..297 297/322 ~ e-167 - (92%) Homo Sapiens (Human), 1..322 297/322 322 aa. (92%) Q9YSY7 LYMPHATIC ENDOTHELIUM- 1..297 294/322 e-165 (91%) SPECIFIC HYALURONAN 1..322 295/322 (91%) RECEPTOR LYVE-1 - Homo Sapiens (Human), 322 aa.

Q99NE4 HYALURONAN RECEPTOR 6..297 202/316 e-106 (63%) PRECURSOR - Mus musculus 6..318 230/316 (71%) (Mouse), 318 aa.

Q98SR5 T CELL ANTIGEN CD44 ~ISOFORM36..116 30/81 (37%)Se-09 B - Anas platyrhynchos 53..132 48/81 (59%) (Domestic duck), 265 aa.

Q90ZL8 T CELL ANTIGEN CD44 ISOFORM36..116 30/81 (37%)Se-09 A - Anas platyrhynchos 53..132 48181 (59%)~
(Domestic duck), 398 aa.

PFam analysis predicts that the NOV 1 a protein contains the domain shown in the Table 1 E.
Table 1E.
Domain Analysis of NOVla Identities! ~

Pfam Doma~isNOVIa Match Region Similarities ~ ~ Expect :'aIue ~

for the Matched Region Xlink 43..104 19/74 (26%) 8.7e-12 43/74 (58%) Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 2A.
Table 2A. NOV2 Sequence Analysis SEQ ID NO: 3 2289 by NOV2a, GTATTCTGGTAGAGGAGGCATCAAGAGTCCTGGGAGGCCGGTGGTAATCATGTAGGCA
CG1OO104- .O1 _CCATGGAAACTGCTATGTGCGTTTGCTGTCCATGTTGTACATGGCAGAGATGTTGTCC
DNA Sequence T~GTTATGCTCCTGTCTGTGCTGCAAGTTCATCTTCACCTCAGAGCGGAACTGCACC

TGCTTCCCCTGCCCTTACAAAGATGAGCGGAACTGCCAGTTCTGCCACTGCACCTGTT
GTGCTGCTGCACAGCCAGCAGCAATCTCAACTGCTACTACTATGAGAGCCGCTGCTGC
CGCAATACCATCATCACTTTCCACAAGGGCCGCCTCAGGAGCATCCATACCTCCTCCA
AGACTGCCCTGCGCACTGGGAGCAGCGATACCCAGGTGGATGAAGTAAAGTCAATACC
AGCCAACAGTCACCTGGTGAACCACCTCAATTGCCCCATGTGCAGCCGGCTGCGCCTG
CACTCATTCATGCTGCCCTGCAACCACAGCCTGTGCGAGAAGTGCCTGCGGCAGCTGC
AGAAGCACGCCGAGGTCACCGAGAACTTCTTCATCCTCATCTGCCCAGTGTGCGACCG
CTCGCACTGCATGCCCTACAGCAACAAGATGCAGCTGCCCGAGAACTACCTGCACGGG
CGTCTCACCAAGCGCTACATGCAGGAGCACGGCTACCTCAAGTGGCGCTTTGACCGCT
CCTCCGGGCCCATCCTCTGCCAGGTCTGCCGCAACAGGCGCATCGCTTACAAGCGCTG
CTGCACCTTCTGCAAGTTCTCTTTCCACAATGGCCACGACACCATTAGCCTCATCGAC
GATATGAAATTGATAATGACCTAATGGAATTCAACATCTTAAAAAACAGCTTTAAAGC
TGACAAGGAGGCAAAGCGAAAAGAGATCAGAAATGGCTTTCTCAAGTTGCGCAGCATT
CTTCAGGAGAAAGAGAAGATCATCATGGAGCAGATAGAGAATCTAGAAGTGTCCAGGC
AGAAGGAAATTGAAAAATATGTGTATGTTAC__AACCATG.AAAGTGAACGAGATGGATGG
TCTGATCGCCTACTCCAAGGAAGCCCTGAAGGAGACTGGCCAGGTGGCATTCCTGCAG
TCAGCCAAGATCCTGGTGGACCAGATCGAGGACGGCATCCAGACCACCTACAGGCCTG
ACCCACAGCTCCGGCTGCACTCAATAAACTACGTGCCCTTGGACTTTGTTGAGCTTTC
CAGTGCCATCCATGAGCTCTTCCCCACAGGGCCCAAGAAGGTACGCTCCTCAGGGGAC
TCCCTGCCCTCCCCCTACCCCGTGCACTCAGAAACAATGATTGCCAGGAAGGTCACTT
TCAGCACCCACAGCCTCGGCAACCAGCACATATACCAGCGAAGCTCCTCCATGTTGTC
CTTCAGCAACACTGACAAGAAGGCCAAGGTGGGTCTGGAGGCCTGTGGGAGAGCCCAG
TCAGCCACCCCCGCCAAACCCACAGACGGCCTCTACACCTACTGGAGTGCTGGAGCAG
CTCTGTGAAGACCCCAGGCCCAATTGTTATCTACCAGACTCTGGTGTACCCAAGAGCT
GCCAAGGTTTACTGGACATGTCCAGCAGAAGACGTGGACTCTTTTGAGATGGAATTCT
TTTAAAGTTAGAGCCATCAATGATAATGGTCCTGGGCAATGGAGTGATATCTGCAAGG
TGGTAACACCAGATGGACATGGGAAGAACCGAGCTAAGTGGGGCCTGCTGAAGAATAT
CCAGTCTGCCCTCCAGAAGCACTTCTGAGCCCCTTCAGAGCAGGAAACAACCTCAGAC
TCATCACAAAGTAGACATATACACACA
ORF Start: ATG at 61 ORF Stop: TGA at 2230 SEQ ID NO: 4 ~ X23 as ~~J at 82771.BkD
NOV2a, METAMCVCCPCCTWQRCCPQLCSCLCCKFIFTSr:~vc:~rc:r~rc:YYII~ERNCQr~CHCTCS

Protein Sequence T~RTGSSDTQVDEVKSIPANSHLVNHLNCPMCSRLRLHSFMLPCNHSLCEKCLRQLQ

SGPILCQVCRNRRIAYKRCITCRLNLCNDCLKAFHSDVAMQDHVFVDTSAEEQDEKIC
IHHPSSRIIEYCRNDNKLLCTFCKFSFHNGHDTISLIDACSERAASLFSAIAKFKAVR
YEIDNDLMEFNILKNSFKADKEAKRKEIRNGFLKLRSILQEKEKIIMEQIENLEVSRQ
PQLRLHSINYVPLDFVELSSAIHELFPTGPKKVRSSGDSLPSPYPVHSETMIARKVTF
STHSLGNQHTYQRSSSMLSFSN'TDKKAKVGLEACGRAQSATPAKPTDGLYTYWSAGAD
SQSVQNSSSF'HNWYSFNDGSVKTPGPIVIYQTLVYPRAAICVYWTCPAEDVDSFEMEFY
SEQ ID NO: 5 X579 by Sequence AATGGAATTCAACATCTTAAAAAACAGCTTTAAAGCTGACAAGGAGGCAAAGCGAAAA

GAGATCAGAAATGGCTTTCTCAAGTTGCGCAGCATTCTTCAGGAGAAAGAGAAGATCA

TCATGGAGCAGATAGAGAATCTAGAAGTGTCCAGGCAGAAGGAAATTGAAAAATATGT

GTATGTTACAACCATGAAAGTGAACGAGATGGATGGTCTGATCGCCTACTCCAAGGAA

GCCCTGAAGGAGACTGGCCAGGTGGCATTCCTGCAGTCAGCCAAGATCCTGCTCGAG

OItF Start: at 1 ~ OIZF Stop: end of sequence SEQ ID NO: 6 193 MW at 22238.2kD
as NOV2b, GSDCLKAFHSDVAMQDHVFVDTSAEEQDEKICIHHPSSRIIEYCRNDNKLLCTFCKFS

198362674 Protein~G~TISLIDACSERAASLFSAIAKFKAVRYEIDNDLMEFNILKNSFKADKEAKRK

Sequence EIRNGFLKLRS~ILQEKEKIIMEQIENLEVSRQKEIEKYVYVTTMFCVNEMDGLIAYSKE

ALKETGQVAFLQSAKILLE

SEQ ID NO: 7 579 by NOV2C, GGATCCGACTGCCTCAAGGCCTTCCACTCGGATGTGGCCATGCAAGACCACGTCTTTG

Sequence CATCATCGAGTACTGCCGCAATGACAACAAATTGCTCTGCACCTTCTGCAAGTTCTCT

TTCCACAATGGCCACGACACCATTAGCCTCATCGACGCCTGCTCCGAGAGGGCCGCCT

CACTCTTCAGCGCCGTCGCCAAGTTCAAAGCAGTCCGATATGAAATTGATAATGACCT

AATGGAATTCAACATCTTAAAAAACAGCTTTAAAGCTGACAAGGAGGCAAAGCGAAAA

GAGATCAGAAATGGCTTTCTCAAGTTGCGCAGCATTCTTCAGGAGAAAGAGAAGATCA

TCATGGAGCAGATAGAGAATCTAGAAGTGTCCAGGCAGAAGGAAATTGAAAAATATGT

GTATGTTACAACCATGAAAGTGAACGAGATGGATGGTCTGATCGCCTACTCCAAGGAA

GCCCTGAAGGAGACTGGCCAGGTGGCATTCCTGCAGTCAGCCAAGATCCTGCTCGAG

ORF Start: at 1 ORF Stop:
end of sequence SEQ ID NO: 8 193 as MW at 22224.21eD

NOV2C, GSDCLKAFIiSDVAMQDHVFVDTSAEEQDEKICIHHPSSRIIEYCRNDNKLLCTFCKFS

198362686 PIOteiri~G~TISI'IDACSERAASLFSAVAKFKAVRYEIDNDLMEFNILKNSFKADKEAKRK

Sequence EIRNGFLKLRSILQEKEKIIMEQIENLEVSRQKEIEKYVYVTTMKVNEMDGLIAYSKE

ALKETGQVAFLQSAKILLE

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 2B.
Table 2B. Comparison of NOV2a against NOV2b and NOV2c.

NOV2a Residues/ Identities/

Protein Sequence Similarities for the Match Residues Matched Region NOV2b 260..451 175/192 (91%) 2..193 178/192 (92%) NOV2c 260..451 174/192 (90%) 2..193 178/192 (92%) Further analysis of the NOV2a protein yielded the following properties shown in Table 2C.
Table 2C. Protein Sequence Properties NOV2a PSort 0.4600 probability located in mitochondria) matrix space; 0.3000 probability located in analysis: microbody (peroxisome); 0.1562 probability located in mitochondria) inner membrane; 0.1562 probability located in mitochondria) intermembrane space SignalP Cleavage site between residues 25 and 26 analysis:
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 2D.
Table 2D. Geneseq Results for NOV2a NOV2a Identities/

Geneseq Protein/Organism/Length Residues/. SimilaritiesExpect [Patent #, for IdentifierDatej Match the Matched Value ResiduesRegion AAM34792Peptide #8829 encoded by 14..113 100/100 (100%)2e-66 probe for measuring placental gene i ..100 l "vG/100 expression - ( l 00 lo) Homo Sapiens, 100 aa. [W0200157272-A2, 09-AUG-2001 ]

ABB41017Peptide #8523 encoded by 14..113 100/100 (140%)2e-66 human foetal liver single exon probe.- 1..100 100/100 (100%) Homo sapiens, 100 aa. [W0200I57277-A2, 2001]

AAM35060Peptide #9097 encoded by 515..620106/106 (100%)2e-56 probe for measuring placental gene 1..106 106/106 (100%) expression -Homo Sapiens, 106 aa. [WO200157272-A2, 09-AUG-2001 ]

AAM74944Human bone marrow expressed515..6201061106 (100%)2e-56 probe encoded protein SEQ ID NO: 1..106 106/106 (100%) Homo Sapiens, 106 aa. [W0200157276-A2, 09-AUG-2001 AAM62140Human brain expressed singleSI5..620~ 106!106 2e-56 exon (100%) proba encoded protein SEQ 1..106 ~ ? Q5/1 ID NO: ~5 (.1.00%) 34245 - Homo sapiens, 106 aa.

[W0200I57275-A2, 09-AUG-2001]

In a BLAST search of public sequence datbases, the NOV2a protein was found to have homology to the proteins shown in the BLASTP data in Table 2E.
Table 2E. Public BLASTP Results for NOV2a Protein NOV2a Identities/

AccessionProtein/Organism/Length Residues/ SimilaritiesExpect for Number Match the Matched Value Residues Portion Q9D2H5 4930486B16RIK PROTEIN -Mus 1..723 629/723 (86%)0.0 musculus (Mouse), 723 aa. 1..723 679/723 (92%) Q90WDl MIDLINE-1 - Gallus gallus 140..52594/411 (22%) Ie-21 (Chicken), 667 aa. 4..402 169/411 (40%) Q9QUS6 MIDLINE 2 PROTEIN - Mus musculus140..52594/411 (22%) 2e-21 (Mouse), 685 aa. 4..402 168/411 (40%) P82458 MIDLINE 1 PROTEIN (RING FINGER140..52594/411 (22%) 3e-21 PROTEIN) - Rattus norvegicus4..402 170/411 (40%) (Rat), 667 aa.

Q9UJV3 RING FINGER PROTEIN 140..52594/411 (22%) 4e-21 (HYPOTHETICAL 77.9 KDA 4..402 167/41 I (39%) PROTEIN) - Homo sapiens (Human), 685 aa.

PFam analysis predicts that the NOV2a protein contains the domains shown in the Table 2F.
Table 2F. Domain Analysis of NOV2a Identities/

Pfam DomainNOV2a Match Similarities Expect Region Value for the Matched Region zf C3HC4 146..191 16/54 (30%) ~ 0.0023 28/54 (52%) zf B box 233..280 10/50 (20%) 0.46 3 1/50 (62%) zf B_box 285..326 14/49 (29%) 0.0033 24/49 (49%) fn3 601..691 17/94 (18%) 0.00093 62/94 (66%) Example 3.
The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.

TGGGGGTCATCGTGGCAGCCGTCCTTGTAACCCTGATTCTCCTGGGAATCTTGGTTTT

TGGCATCTGGTTTGCCTATAGCCGAGGCCACTTTGACAGAACAAAGAAAGGGACTTCG

AGTAAGAAGGTGATTTACAGCCAGCCTAGTGCCCGAAGTGAAGGAGAATTCAAACAGA

CCTCGTCATTCCTGGTGTGAGCCTGGTCGGCTCACCGCCTATCATCTGCATTTG

ORF Start: ATG at 39 ORF Stop:
TGA
at 714 SEQ ID NO: 10 225 as MW at 24525.6kD

NOV3a, MGTKAQVERKLLCLFILAILLCSLALGSVTVHSSEPEVRIPENNPVKLSCAYSGFSSP

CG100114-O1 R~~~QGDTTIGNRAVLTCSEQDGSPPSEYTWFKDGIVMPTNPKSTRAFSNSSYVL

Protein Sequence NPTTGELVFDPLSASDTGEYSCEARNGYGTPMTSNAVRMEAVERNVGVIVAAVLVTLI

LLGILVFGIWFAYSRGIiFDRTKKGTSSKKVIYSQPSARSEGEFKQTSSFLV

Further analysis of the NOV3a protein yielded the following properties showwin Table 3B.
Table 3B. Protein Sequence Properties NOV3a PSort 0.4600 probability located in plasma membrane; 0.1000 probability located in analysis: endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic r_eticulum (lumen); 0.1000 probability located in outside .
SignalP Cleavage site between residues 28 and 29 analysis:
A search of the NOV3a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3C.
Table 3C. Geneseq Results for NOV3a NOV3a Identifies/

Geneseq Protein/Organism~/Lengtli Residues/SipnilaritiesExpect [Patent #, fo:

IdentifierDate]' Matc'a the l~atslDedVal;:c "

ResiduesRegion ABB72215Human protein isolated from70..225 156/156 (100%)4e-86 skin cells SEQ ID NO: 331 - Homo sapiens,144..299156/156 (100%) aa. [W0200190357-A1, 29-NOV-2001]

ABB72150Human protein isolated from70..225 156/156 (100%)4e-86 skin cells ~

SEQ ID NO: 189 - Homo sapiens,144..299156/156 (100%) aa. [W0200190357-A1, 29 NOV-2001]

AAB53086Human angiogenesis-associated70..225 156/156 (100%)4e-86 protein PR030I, SEQ ID N0:119 - 144..299156/156 (100%) Homo sapiens, 299 aa. [W0200053753-A2, SEP-2000]

AAB56015Skin cell protein, SEQ ID 70..225 156/156 (100%)4e-86 NO: 331-Homo Sapiens, 299 aa. [W0200069884-144..299156/156 (100%) ~ A2, .
23-NOV-2000]

AAB55950 Skin cell protein, SEQ ID NO: 189 - 70..225 156/156 (100%) 4e-86 Homo sapiens, 299 aa. [W0200069884- 144..299 156/156 (100%) A2, 23 NOV-2000]
In a BLAST search of public sequence datbases, the NOV3a protein was found to have homology to the proteins shown in the BLASTP data in Table 3D.
Table 3D. Public BLASTP Results for NOV3a NOV3a Identities/

Protein Residues/Similarities Expect for AccessionProtein/Organism/Length Match the Matched Value Number ResiduesPortion Q9YSB2 JUNCTION ADHESION MOLECULE I ..225 198/279 (70%)3e-98 -Homo sapiens (Human), 259 1..259 202/279 (71%) aa.

Q9Y624 functional adhesion molecule70..225 156/156 (100%)9e-86 1 precursor (JAM) (Platelet adhesion 144..299156/156 (100%) molecule 1) (PAM-1 ) (Platelet F 11 receptor) - Homo sapiens (Human), 299 aa.

Q9XT56 functional adhesion molecule70..225 1191156 (76%)1e-66 l precursor (JAM) - Bos taurus (Bovine),143..298138/156 (88%) 298 aa.

Q9JHY1 JUNCTIONAL ADHESION 70..225 125/158 (79%)2e-65 MOLECULE JAM - Rattus norvegicus143..300140/158 (88%) (Rat), 300 aa.

Q9JKD5 JUNCTIONAL ADHESION 70..225 125/158 (79%)2e-65 MOLECULE - Rattus norvegicus16..173 140/158 (88%) (Rat), 173 as (fragment).

PFam analysis predicts that the NOV3a protein contains the domains shown in the Table ~3E.
Table 3E. Domain Analysis of NOV3a Identities/
Pfam Domain NOV3a Match Region Similarities Expect Value for the Matched Region Ig 43..67 8/27 (30%) 0.1 S
21/27 (78%) 1g 72..140 15/71 (21%) 1.2e-08 52/71 (73%) .

Example 4.
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.
Table 4A. NOV4 Sequence Analysis SEQ ID NO: 11 1545 by NOV4a, _CATGAGTGTGGTGCTGGTGCTACTTCCTACACTGCTGCTTGTTATGCTCACGGGTGCT

DNA SequenceATGCTTTCGCAGATATCCCTGAGAACATTTCTGGAGGGTCACAAGGCTTATCATTAAG

GTTCAACAGCATTCAGAAGCTCAAATCCAATCAGTTTGCCGGCCTTAACCAGCTTATA

TGGCTTTATCTTGACCATAATTACATTAGCTCAGTGGATGAAGATGCATTTCAAGGGA

TCCGTAGACTGAAAGAATTAATTCTAAGCTCCAACAAAATTACTTATCTGCACAATAA

AACATTTCACCCAGTTCCC"~-~AT~mCCGCAAmCTGC',p~CCTCTCCTACAATAAGCTTCAG

ACATTGCAATCTGAACAATTTAAAGGCCTTCGGAAACTCATCATTTTGCACTTGAGAT

CTAACTCACTAAAGACTGTGCCCATAAGAGTTTTTCAAGACTGTCGGAATCTTGATTT

TTTGGATTTGGGTTACAATCGTCTTCGAAGCTTGTCCCGAAATGCATTTGCTGGCCTC

TTGAAGTTAAAGGAGCTCCACCTGGAGCACAACCAGTTTTCCAAGATCAACTTTGCTC

ATTTTCCACGTCTCTTCAACCTCCGCTCAATTTACTTACAATGGAACAGGATTCGCTC

CATTAGCCAAGGTTTGACATGGACTTGGAGTTCCTTACACAACTTGGATTTATCAGGG

AATGACATCCAAGGAATTGAGCCGGGCACATTTAAATGCCTCCCCAATTTACP~AAAAT

TGAATTTGGATTCCAACAAGCTCACCAATATCTCACAGGAAACTGTCAATGCGTGGAT

ATCATTAATATCCATCACATTGTCTGGAAATATGTGGGAATGCAGTCGGAGCATTTGT

CCTTTATTTTATTGGCTTAAGAATTTCAAAGGAAATAAGGAAAGCACCATGATATGTG

CGGGACCTAAGCACATCCAGGGTGAAAAGGTTAGTGATGCAGTGGAAACATATAATAT

CTGTTCTGAAGTCCAGGTGGTCAACACAGAAAGATCACACCTGGTGCCCCAAACTCCC

CAGAAACCTCTGATTATCCCTAGACCTACCATCTTCAAACCTGACGTCACCCAATCCA

CCTTTGAAACACCAAGCCCTTCCCCAGGGTTTCAGATTCCTGGCGCAGAGCAAGAGTA

TGAGCATGTTTCATTTCACAAAATTATTGCCGGGAGTGTGGCTCTCTTTCTCTCAGTG

GCCATGATCCTCTTGGTGATCTATGTGTCTTGGAAACGCTACCCAGCCAGCATGAAAC

AACTCCAGCAACACTCTCTTATGAAGAGGCGGCGGAAAAAGGCCAGAGAGTCTGAAAG

ACAAATGAATTCCCCTTTACAGGAGTATTATGTGGACTACAAGCCTACAAACTCTGAG

ACCATGGATATATCGGTTAATGGATCTGGGCCCTGCACATATACCATCTCTGGCTCCA

GGGAATGTGAGGTATGAACCATGATCCTCCTAAAAGC

ORF Start: ATG at 2 ORF Stop: TGA
at 1523 SEQ ID NO: 12 507 MW at 57899.I1cD
as NOV4a, MSVVLVLLPTLLLVMLTGAQRACPKNCRCDGKIVYCESHAFADIPENISGGSQGLSLR

CG100619-O1 ~SIQ~KSNQFAGLNQLIWLYLDHNYISSVDEDAFQGIRRLKELILSSNKITYLHNK

Protein SequenceT~PVPNLRNLDLSYNKLQTLQSEQFKGLRKLIILHLRSNSLKTVPIRVFQDCRNLDF

LDLGYNRLRSLSRNAFAGLLKLKELHLEHNQFSKINFAHFPRLFNLRSIYLQWNRIRS

ISQGLTWTWSSLHNLDLSGNDIQGIEPGTFKCLPNLQKLNLDSNKLTNISQETVNAWI

SLISITLSGNMWECSRSICPLFYWLKNFKGNKESTMICAGPKHIQGEKVSDAVETYNI

CSEVQVVNTERSHLVPQTPQKPLIIPRPTIFKPDVTQSTFETPSPSPGFQIPGAEQEY

EHVSFHKIIAGSVALFLSVAMILLVIYVSWKRYPASMKQLQQHSLMKRRRKKARESER

QMNSPLQEYYVDYKPTNSETMDISVNGSGPCTYTISGSRECEV

SEQ ID NO: 13 1194 by NOV4b, GGTACCCAGAGAGCTTGCCCAAAGAACTGCAGATGTGATGGCAAAATTGTGTACTGTG

DNA

Sequence ATTAAGGTTCAACAGCATTCAGAAGCTCAAATCCAATCAGTTTGCCGGCCTTAACCAG

CTTATATGGCTTTATCTTGACCATAATTACATTAGCTCAGTGGATGAAGATGCATTTC

AAGGGATCCGTAGACTGAAAGAATTAATTCTAAGCTCCAACAAAATTACTTATCTGCA

CAATAAAACATTTCACCCAGTTCCCAATCTCCGCAATCTGGACCTCTCCTACAATAAG

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 4B.
Table 4B. Comparison of NOV4a against NOV4b.
Protein Sequence NOV4a Residues/ Identities/
Match Residues Similarities for the Matched Region NOV4b 18..414 384/397 (96%) 1..397 385/397 (96io) Further analysis of the NOV4a protein yielded the following properties shown in Table 4C.
Table 4C. Protein Sequence Properties NOV4a Psort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400 probability analysis: located in plasma membrane; 0.4600 probability located in Golgi body; 0.1000 probability located in endoplasmic reticulum (lumen) SignalP Cleavage site between residues 20 and 21 analysis:
A search of the NOV4a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4D.

Table 4D. Geneseq Results for NOV4a NOV4a Identities/
Geneseq Protein/Organism/Length [Patent Expect #, IdentifierDate] Residues/SimilaritiesValue for Match the Matched Residues Region AAB66201Protein of the invention 2..507 327/512 0.0 #113 - (63%) Unidentified, 513 aa. [W0200078961-A1,13..513 403/512 (77%) 28-DEC-2000]

AAB87587Human PR01693 - Homo Sapiens,2..507 327/512 0.0 513 aa. (63%) [W0200116318-A2, 08-MAR-2001]13..513 403/512 (77%) AAU12439Human PRO1693 polypeptide 2..507 327/512 0.0 sequence - (63%) Homo Sapiens, S 13 aa. [W0200140466-13..513 403/512 (77%) A2, 07-JUN-2001 ]

AAY99452Human PRO1693 (UNQ803) amino~ 2..507 327/512 0.0 acid (63%) sequence SEQ ID N0:385 - ~ 13..513403/512 Homo (77%) Sapiens, 513 aa. [W0200012708-A2, MAR-2000]

AAB65236Human PR01309 (UNQ675) protein~ 3..507 244/514 e-135 (47%) sequence SEQ ID N0:278 - 20..522 339/514 Homo (65%) sapiens, 522 aa. [W0200073454-Al, DEC-2000]

In a BLAST search of public sequence datbases, the NOV4a protein was found to have homology to the proteins shown in the BLASTP data in Table 4E.
Table 4E. Public BLASTP Results for Nv~ V :a Protein ' NOV4a Identities/

AccessionProtein/Organism/Length Residues/SimilaritiesExpect for Number Match the Matched Value ResiduesPortion Q9BGP6 HYPOTHETICAL 65.9 IRDA PROTEIN2..507 326/512 (63%)0.0 - Macaca fascicularis (Crab13..513 403/512 (78%) eating macaque) (Cynomolgus monkey), aa.

Q95KI8 HYPOTHETICAL 65.9 KDA PROTEIN2..507 324/512 (63%)0.0 - Macaca fascicularis (Crab13..513 402/512 (78%) eating macaque) (Cynomolgus monkey), aa.

Q96DN1 CDNA FLJ32082 FIS, CLONE OCBBF2000231,3..507 244/514 e-(47%) WEAKLY SIMILAR TO PHOSPHOLIPASE A2 20..522340/514 135 (65%) INHIBITOR SUBUNIT B PRECURSOR - Homo sapiens (Human), 522 aa.

Q9H9T0 CDNA FLJ12568 FIS, CLONE NT2RM4000857,301..507207/207 e-(100%) WEAKLY SIMILAR TO LEUCINE-RICH ALPHA- 1..207 207/207 120 (100%) 2-GLYCOPROTEIN (HYPOTHETICAL 23.6 KDA

PROTEIN) - Homo Sapiens (Human), 207 aa.

043300 KIAA0416 PROTEIN - Homo Sapiens 1..507 227/5/ 1 e-(Human), 516 (44%) aa. 20..516327/511 118 (63%) PFam analysis predicts that the NOV4a protein contains the domains shown in the Table 4F.
Table 4F. Domain Analysis of NOV4a Identities/

Pfam DomainNOV4a Match Similarities Expect Region Value for the Matched Region LRRNT 22..49 9131 (29%) 0.043 18/31 (58%) LRR 75..98 7125 (28%) 0.068 20/25 (80%}

LRR 99..122 9/25 (36%) 0.33 18/25 (72%) LRR 123..146 12/25 (48%) 0.0015 21/25 (84%) ' LRI~ ~ 147..170 7/25 (28%) 0.48 20/25 (80%) LRR 171..194 11/25 (44%) 0.014 20/25 (80%) LRR 243..266 10/25 (40%) 0.0004 20125 (80%) LRRCT 300..350 11/55 (20%) 0.25 34/55 (62%) Example 5.
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table SA.

Table SA. NOVS Sequence Analysis SEQ ID NO: I 5 743 by NOVSa, GTGCCAGCGCGGCGTGGGCCTCGGTCTGCGGCCATGGGGGTGTCCTCGCGGCTGCTGC

DNA

Sequence TCAATACTTCGAGCTAAAGCACCTTTCCAGCGGGGACCTGCTCCGGGACAACATGCTG

CGGGGCGCAGAAATTGGCCTGTTAGCCAAGGCTTTCATTGACCAAGGGAAACTCATCC

CAAATGATGTCATCTTGGGCGTGGCCCTTCAGGAACTGCAAAATCTCACCCAGTCTAG

GCTGTTGGATAGTTTTCCAAGGACACTTCCACAGGCAGAAGCCCTAGATAAAGCTGAT

CAGACCGACACAGTGATTAACCTGAATATGTCCTTTGAGGTCATTAAACAACGCCTTA

CTGCTCACTGGATTCATCTCACCAATGGCCAAGTCTACAACATTGGATTCAACCCTCC

CACAACTGTGGGCATTGATGCTCTGACAGGGGAGCCGCTCATTCAGCGTGAGGATGAT

AAACCAGAGATGGTTATCAAGAGACTAAAGGCTTATGAAGCCAAACAAAGCCAGTCCT

GGACTATTACCAGAP.AAAAAGCGGTGTTGGAAACATTCTCCAGAACAGAAACCAACAA

GATTTGGCCCTGTGGATATGCTTTCCTCCAAACTGACGTTCCTCAAACAAGCCAGGAA

GCTTCAGTTACTCTATAAGGAGAAATGTGTGGAACTATTAGTAGTAA

ORF Start: ATG at 34 ORF Stop: TAA
at 712 SEQ ID NO: 16 226 MW at 25110.6kD
as NOVSa, MGVSSRLLRWIMGAPGSGKGTVSSRITQYFELKHLSSGDLLRDNMLRGAEIGLLAKA

Protein SequenceFEVIKQRLTAHWIFiLTNGQVYNIGFNPPTTVGIDALTGEPLIQREDDKPEMVIKRLKA

YEAKQSQSWTITRKKAVLETFSRTETNKIWPCGYAFLQTDVPQTSQEASVTL

Further analysis of the NOVSa protein yielded the following properties shown in Table SB.
Table SB. Protein Sequence Properties NOVSa PSort 0.3600 probability located in mitochondria) matrix space; 0.3000 probability located analysis: in microbody (peroxisome); 0.2224 probability located in lysosome (lumen); 0.0000 probability located in endoplasmic reticulum (membrane) SignalP No Known Signal Sequence Predicted analysis:
A search o...f .t>ae NOVSa protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table SC.
Table SC. Geneseq Results for NOVSa NOVSa Identities/
Geneseq Protein/Organism/Length Residues/ Similarities for the Expect Value Identifier [Patent #, Date) Match Matched Region Residues AAW81101 Human mitochondria) adenylate kinase 1..225 177/226 (78%) 1e-92 protein - Homo Sapiens, 227 ad. 1..226 191/226 (84%) [W09844124-A1, 08-OCT-1998]

AAB85885 Human adenylate kinase 3 1..225 177/226 (78%) (AK3)-like ~ 1 e-92 protein - Homo Sapiens, 227 aa. 1..226 191/226 (84%) ['1V0200109346-A1, 08-FEB-2001) AAB93487 Human protein sequence SEQ 1..225 177/226 (78%) ID 1 e-92 N0:12786 - Homo Sapiens, 227 aa. 1..226 191/226 (84%) [EP1074617-A2, 07-FEB-2001) AAB93066 Human protein sequence SEQ 1..225 177/226 (78%) ID 1 e-92 N0:11883 - Homo Sapiens, 227 aa. 1..226 191/226 (84%) [EP 1074617-A2, 07-FEB-2001 ) AAB92887 Human protein sequence SEQ 1..225 177/226 (78%) ID 1 e-92 N0:11492 - Homo Sapiens, 227 aa. 1..226 191/226 (84%) [EP1074617-A2, 07-FEB-2001) In a BLAST search of public sequence datbases, the NOVSa protein was found to have homology to the proteins shown in the BLASTP data in Table SD.
Table SD. Public BLASTP Results for NOVSa Protein NOVSa Identities/.

Accession Protein/Organism/Length Residues)SimilaritiesEzpect for Number Match the MatchedValue ResiduesPortion Q9NPB4 CDNA FLJI 1089 FIS, CLONE 1..225 177/226 3e-92 (78%) PLACE1005305, HIGHLY SIMILAR TO 1..226 191/226 (84%) GTP:AMP PHOSPHOTRANSFERASE

MITOCHONDRIAL (EC 2.7.4.10) (CDNA

FLJ10691 FIS, CLONE NT2RP3000359, HIGHLY SIMILAR TO GTP:AMP

PHOSPHOTRANSFERASE

MITOCHONDRIAL) (CDNA FLJ14628 FIS, CLONE NT2RP2000329, HIGHLY SIMILAR

_ _ TO GTP:AMP PHOSPHOTRANSFERASE

MITOCHONDRIAL) (HYPOTHETICAL

25.6 KDA PROTEIN) - Homo sapiens . (Human), 227 aa.

A34442 nucleoside-triphosphate--adenylate1..225 170/226 3e-90 kinase (EC (75%) 2.7.4.10) 3, mitochondria) - bovine, 1..226 188/226 227 aa. (82%) Q9D7Z1 ADENYLATE KINASE 3 ALPHA LIKE 1..225 172/226 4e-90 - (76%) ~

Mus musculus (Mouse), 227 aa. 1..226 189/226 (83%) Q9DBM5 ADENYLATE KINASE 3 ALPHA LIKE 1..225 171/226 1e-89 - . (75%) Mus musculus (Mouse), 227 aa. 1..226 189/226 (82%) P08760 GTP:AMP phosphotransferase mitochondria!2..225 169/225 1e-89 (75%) (EC 2.7.4.10) (AK3) - Bos taurus (Bovine),1..225 187/225 (83%) 226 aa.

PFam analysis predicts that the NOVSa protein contains the domain shown in the Table SE.

Table 5E. Domain Analysis of NOVSa Identities/
Pfam Domain NOVSa Match Region Similarities Expect Value for the Matched Region adenylatekinase 12..178 77/176 (44%) 1.2e-65 137/176 (78%) Example 6.
The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.
Table 6A. NOV6 Sequence Analysis SEQ ID NO: 17 X2153 by NOV6a, GATGCTGGCACTTACATGTGTGTGGCCCAGAACCCGGCTGGTACAGCCTTGGGCAAAA
CG56914-OI DNA T~GTTAAATGTCCAAGTTCCTCCAGTCATTAGCCCTCATCTAAAGGAATATGTTAT
Sequence TGCTGTGGACAAGCCCATCACGTTATCCTGTGAAGCAGATGGCCTCCCTCCGCCTGAC
ATTACATGGCATAAAGATGGGCGTGCAATTGTGGAATCTATCCGCCAGCGCGTCCTCA
GCTCTGGCTCTCTGCAAATAGCATTTGTCCAGCCTGGTGATGCTGGCCATTACACGTG
CATGGCAGCCAATGTAGCAGGATCAAGCAGCACAAGCACCAAGCTCACCGTCCATGTA
CCACCCAGGATCAGAAGTACAGAAGGACACTACACGGTCAATGAGAATTCACAAGCCA
TTCTTCCATGCGTAGCTGATGGAATCCCCACACCAGCAATTAACTGGAAAAAAGACAA
TGTTCTTTTAGCTAACTTGTTAGGAAAATACACTGCTGAACCATATGGAGAACTCATT
TTAGAAAATGTTGTGCTGGAGGATTCTGGCTTCTATACCTGTGTTGCTAACAATGCTG
CAGGTGAAGATACACACACTGTCAGCCTGACTGTGCATGTTCTCCCCACTTTTACTGA
TTCAGGTACTTATGTGTGCACCGCAGAGAACAGCGTTGGCTTTGTGAAGGCAATTGGA
TTTGTTTATGTGAAAGAACCTCCAGTCTT~..AAAGGTGATTATCCTTCTAACTGGATTG
AACCACTTGGTGGGAATGCAATCCTGAATTGTGAGGTGAAAGGAGACt~_C'c:,P~'Ce'~'1~AC
CATCCAGTGGAACAGAAAGGGAGTGGATATTGAAATTAGCCACAGAATCCGGCAACTG
GGCAATGGCTCCCTGGCCATCTATGGCACTGTTAATGAAGATGCCGGTGACTATACAT
GTGTAGCTACCAATGAAGCTGGGGTGGTGGAGCGCAGCATGAGTCTGACTCTGCAAAG
TCCTCCTATTATCACTCTTGAGCCAGTGGAAACTGTTATTAATGCTGGTGGCAAAATC
ATATTGAATTGTCAGGCAACTGGAGAGCCTCAACCAACCATTACATGGTCCCGTCAAG
GGCACTCTATTTCCTGGGATGACCGGGTTAACGTGTTGTCCAACAACTCATTATATAT
TGCTGATGCTCAGAAAGAAGATACCTCTGAATTTGAATGCGTTGCTCGAAACTTAATG
CTTGGGGAACATGCAGCGAAAGTTGTGGGAAAGGTACTCAGACAAGAGCAAGACTTTG
TAATAACCCACCACCAGCGTTTGGTGGGTCCTACTGTGATGGAGCAGAAACACAGATG
CAAGTTTGCAATGAAAGAAATTGTCCAATTCATGGCAAGTGGGCGACTTGGGCCAGTT
GGAGTGCCTGTTCTGTGTCATGTGGAGGAGGTGCCAGACAGAGAACAAGGGGCTGCTC
CGACCCTGTGCCCCAGTATGGAGGAAGGAAATGCGAAGGGAGTGATGTCCAGAGTGAT
TTTTGCAACAGTGACCCTTGCCCAAGTGAGTGTTGGAAATACCCATGGTAACTGGAGT
1~~

CCTTGGA

ORF Start: ATG at 16 r ORF Stop: TAA at 2137 SEQ ID NO: I8 707 as MW at 76557.7kD

NOV6a, MCVAQNPAGTALGKIKLNVQVPPVISPHLKEYVIAVDKPITLSCEADGLPPPDITWHK

Protein SequenceSTEGHYTVNENSQAILPCVADGIPTPAINWKKDNVLLANLLGKYTAEPYGELILENW

LEDSGFYTCVANNAAGEDTHTVSLTVHVLPTFTELPGDVSLNKGEQLRLSCKATGIPL

PKLTWTFNNNIIPAHFDSVNGHSELVIERVSKEDSGTYVCTAENSVGFVKAIGFVYVK

EPPVFKGDYPSNWIEPLGGNAILNCEVKGDPTPTIQWNRKGVDIEISHRIRQLGNGSL

AIYGTVNEDAGDYTCVATNEAGVVERSMSLTLQSPPIITLEPVETVINAGGKIILNCQ

ATGEPQPTITWSRQGHSISWDDRVNVLSNNSLYIADAQKEDTSEFECVARNLMGSVLV

RVPVIVQVHGGFSQWSAWRACSVTCGKGIQKRSRLCNQPLPANGGKPCQGSDLEMRNC

QNKPCPVDGSWSEWSLWEECTRSCGRGNQTRTRTCNNPSVQHGGRPCEGNAVEIIMCN

IRPCPVHGAWSAWQPWGTCSESCGKGTQTRARLCNNPPPAFGGSYCDGAETQMQVCNE

RNCPIHGKWATWASWSACSVSCGGGARQRTRGCSDPVPQYGGRKCEGSDVQSDFCNSD

PCPSECWKYPW

SEQ ID NO: 19 15660 by NOV6b, GATTAGTGGCATAAACTGTAGGTCAGCTGGTGGAGGCAAGCCAGCAAGGGGCTTCATG
~

DNA

Sequence CTTCAGCCGCTAAGCCGAGAAGATCTGGGAAGGAGTCAGTCAGAGAGCCTTGGGCCAG

AGTTCCAGGGGCTCTGGGAGTGGCTGCCAGAAAATACCAGAAAATGAAAGGAATTGAA

ATTAAGAGAAGGGAGAGATTGAAGTGTGGCGCCAAGATTGAAAGGAGAAAGAGGTTGA

AGGATAGGGAGGTTGGAGAAGAGAGTAAAAAGAGGCCACTTACTGGATTTGAAATTGA

ACCACCCAAAGTCACTGTGATGCCCAAGAATCAGTCTTTCACAGGAGGGTCTGAGGTC

TCCATCATGTGTTCTGCAACAGGTTATCCCAAACCAAAGATTGCCTGGACCGTTAACG

ATATGTTTATCGTGGGTTCACACAGGTATAGGATGACCTCAGATGGTACCTTATTTAT

CAAAAATGCAGCTCCCAAAGATGCAGGGATCTATGGTTGCCTAGCAAAAGCCCCTAAG

TTGATGGTAGTTCAGAGTGAGCTCTTGGTTGCCCTTGGGGATATAACCGTTATGGAAT

GCAAAACCTCTGGTATTCCTCCACCTCAAGTTAAATGGTTCAAAGGAGATCTTGAGTT

GAGGCCCTCAACATTCCTCATTATTGACCCTCTCTTGGGACTTTTGAAGATTCAAGAA

ACACAAGATCTGGATGCTGGCGATTATACCTGTGTAGCCATCAATGAGGCTGGAAGAG

CAACTGGCAAGATAACTCTGGATGTTGGCTCACCTCCAGTTTTCATACAAGAACCTGC

TGATGTGTCTATGGAAATTGGCTCAAATGTGACATTACCTTGTTATGTTCAGGGTTAT

CCAGAACCAACAATCAAATGGCGAAGATTAGACAACATGCCAATTTTCTCAAGACCTT

TTTCAGTTAGTTCCATCAGCCAACTAAGAACAGGAGCTCTCTTTATTTTAAACTTATG

GGCAAGTGATAAAGGAACCTATATTTGTGAAGCTGAAAACCAGTTTGGAAAGATCCAG

TCAGAGACAACAGTAACAGTGACCGGACTTGTTGCTCCACTTATTGGAATCAGCCCTT

CAGTGGCCAATGTTATTGAAGGACAGCAGCTTACTTTGCCCTGTAcTCTGTTAGCTGG

AAATCCCATTCCAGAACGTCGGTGGr"~TTAAGA.'vT iy:AC~c:~I~ATG~iutsC'1'(:CAAAAhTCCT

TACATCACTGTGCGCAGTGATGGGAGCCTCCATATTGAAAGAGTTCAGCTTCAGGATG

GTGGTGAATATACTTGTGTGGCCAGTAACGTTGCTGGGACCAATAACAAAACTACCTC

TGTGGTTGTGCATGTTCTGCCAACCATTCAGCATGGGCAGCAGATACTCAGTACAATT

GAAGGCATTCCAGTAACTTTACCATGCAAAGCAAGTGGAAATCCCAAACCGTCTGTCA

TCTGGTCCAAGGTAAATGATACATCTAGTTATATTTCCTGAAGAGCAGAGTGTGAAGT

TCACCTGCAAGTTATCCCTAGTCTTGAGCAGGAGGCTCAGGAGTGGGGCATGGAAAGA

AGATAAGTTAATAAAGGATTTCCTATGTGGCTGGACAGATGTGCTAGGAACCCTCCAA

GAAACCATATAGATGCACCTCAGAAGGCTCCCTCGGCTTTTCGCCGTGTTTTGCAGAA

AGGAGAGCTGATTTCAACCAGCAGTGCTAAGTTTTCAGCAGGAGCTGATGGTAGTCTG

TATGTGGTATCACCTGGAGGAGAGGAGAGTGGGGAGTATGTCTGCACTGCCACCAATA

CAGCCGGCTACGCCAAAAGGAAAGTGCAGCTAACAGTCTATGTAAGGCCCAGAGTGTT

TGGAGATCAACGAGGACTGTCCCAGGATAAGCCTGTTGAGATCTCCGTCCTTGCAGGG

GAAGAGGTAACACTTCCATGTGAAGTGAAGAGCTTACCTCCACCCATAATTACTTGGG

CCAAAGAAACCCAGCTCATCTCACCGTTCTCTCCAAGACACACATTCCTCCCTTCTGG

TTCAATGAAGATCACTGAAACCCGCACTTCAGATAGTGGGATGTATCTTTGTGTTGCC

ACAAATATTGCTGGGAATGTGACTCAGGCTGTCAAATTAAATGTCCATGTTCCTCCAA

AGATACAGCGTGGACCTAAACATCTCAAAGTCCAAGTTGGTCAAAGAGTGGATATTCC

ATGTAATGCTCAAGGGACTCCTCTTCCTGTAATCACCTGGTCCAAAGGTGGAAGCACT

ATGCTGGTTGATGGAGAGCACCATGTTAGCAATCCAGACGGAACTTTAAGCATCGACC

TGATGAAACAGAGATAACGCTACATGTCCAAGAACCACCCACAGTGGAAGATCTAGAA
CCTCCATATAACACTACTTTCCAAGAAAGAGTGGCCAATCAACGCATTGAATTTCCAT
GACAGGCAGAGAGCCTGGCATTTCTATCTTGGAAGATGGCACATTGCTGGTTATTGCT
TCTGTTACACCCTATGACAATGGGGAGTACATCTGTGTGGCAGTCAATGAAGCTGGAA
CCACAGAAAGAAAATATAACCTCAAAGTCCATGTTCCTCCAGTAATTAAAGATAAAGA
TCAGCTGACCAATCTCTTCTGTGAAGTG
GAAGGCACTCCATCTCCCATCATTATGTGGTATAAAGATAATGTCCAGGTGACTGAAA
GCAGCACTATTCAGACTGTGAACAATGGGAAGATACTGAAGCTCTTCAGAGCCACTCC
TACTTTAACATTGATGTGCTAGGTACCAACTTCCCAAATGAAGTCTCAGTTGTCCTCA
GTTCAAAGATGGCAATATTAAAGGAGGAAATGTCACCACAGACATATCAGTATTGATC
TGTGAAACACGGGGACTTCCAATGCCTGCCATTACTT
GGTATAAGGACGGGCAGCCAATCATGTCCAGCTCACAAGCACTTTATATTGATAAAGG
ACAATATCTTCATATTCCTCGAGCACAGGTCTCTGATTCAGCAACATATACGTGTCAC
GTAGCCAATGTTGCTGGAACTGCTGP~P.AAATCATTCCATGTGGATGTCTATGTTCCTC
CAATGATTGAAGGCAACTTGGCCACGCCTTTGAATAAGCAAGTAGTTATTGCTCATTC
TCTGACACTGGAGTGCAAAGCTGCTGGAAACCCTTCTCCCATTCTCACCTGGTTGAAA
GATGGTGTACC~T~GuGAAiaGCTA.~~uGACt~.ATATCCGCATAGAAGCTGG'TGGt~AAGAAAG
TCGAAATCATGAGTGCCCAAGAAATTGATCGAGGACAGTACATATGCGTGGCTACCAG
TGTGGCAGGAGAAAAGGAAATCAAATATGAAGTTGATGTCTTGGTGCCACCAGCTATA
GAAGGAGGAGATGAAACATCTTACTTCATTGTGATGGTTAATAACTTACTGGAGCTAG
AATTGATGAAAGGGATGGATTCAAGATTTTATTAAATGGACGCAAACTGGTTATTGCT
CAGGCTCAAGTGTCAAACACAGGCCTTTATCGGTGCATGGCAGCAAATACTGCTGGAG
CCTTTCTGAGAGAGTTGTGGTAAAATACAAGCCTGTCGCCTTGCAGTGCATAGCCAAT
GGGATTCCAAATCCTTCCATTACATGGTTAAAAGATGACCAGCCTGTGAACACTGCCC
AAGGAAACCTTAAAATACAGTCTTCTGGTCGAGTTCTACAAATTGCCAAAACCCTGTT
GGAAGATGCTGGCAGATACACATGTGTGGCTACCAACGCAGCTGGAGAAACACAACAG
CACATTCAACTGCATGTTCATGAACCACCTAGTCTGGAAGATGCTGGAAAAATGCTGA
ATGAGACTGTGTTGGTGAGCAACCCTGTACAGCTGGAGTGTAAGGCAGCTGGAAATCC
TGTGCCTGTTATTACATGGTACAAAGATAATCGTCTACTCTCAGGTTCCACCAGCATG
ACTTTCTTGAACAGAGGACAGATCATTGATATTGAAAGTGCCCAGATCTCAGATGCTG
GCATATATAAATGCGTGGCCATCAACTCAGCTGGAGCTACAGAGTTATTTTACAGTCT
GCAAGTTCATGTGGCCCCATCAATTTCTGGCAGCAATAACATGGTGGCAGTGGTGGTT
AATAACCCGGTGAGGTTAGAATGTGAAGCCAGAGGTATTCCTGCCCCAAGTCTGACCT
GGTTGAAAGATGGGAGTCCTGTTTCTAGTTTTTCTAATGGATTACAGGTTCTCTCTGG
TGGTCGAFiTCCTAGCATTGACCAGTGCACAAATCAGCGACACAGi AAUt~TAC Av:.i'Pr(' GTGGCAGTGAATGCTGCTGGAGAAAAGCAAAGGGACATTGACCTCCGAGTATATGTTC
CGCCAAATATTATGGGAGAAGAACAGAATGTCTCTGTCCTCATTAGCCAAGCTGTGGA
CACCCCTTGCTGAAGAAACCAGGCCTCAGTATATCTGAAAATAGAAGTGTGTTAAAGA
TTGAAGATGCTCAGGTTCAAGACACTGGTCGTTACACTTGTGAAGCAACAAATGTTGC
TGGAAAAACTGAAAA:9AACTACAATGTCAACATTTGGGTCCCCCCAAATATTGGTGGT
TCTGATGAACTTACTCAACTTACAGTCATTGAAGGGAATCTCATTAGTCTGTTGTGTG
GACTGATTCCATGGGGCGAGTTAGAATTTTATCTGGGGGCAGGCAATTACAAATTTCA
ATTGCTGAAAAGTCTGATGCAGCACTCTATTCATGTGTGGCGTCGAATGTTGCTGGGA
CAGCCACCCTACTGAAATTATTGTGACCCGAGGGAAGAGTATCTCCTTGGAGTGTGAG
GTGCAGGGTATTCCACCACCAACAGTGACCTGGATGAAAGATGGCCACCCCTTGATCA
AGGCAAAGGGAGTAGAAATACTGGATGAAGGTCACATCCTTCAGCTGAAGAACATTCA
TGTATCTGACACAGGCCGTTATGTGTGTGTTGCTGTGAATGTAGCAGGAATGACTGAC
AAAAAATATGACTTAAGTGTCCATGGAGGCAGGATGCTACGGCTGATGCAGACCACAA
TGGAAGATGCTGGCCAATATACTTGCGTTGTAAGGAATGCAGCTGGTGAAGAAAGAAA
AATCTTTGGGCTTTCAGTATTAGTACCACCTCATATTGTGGGTGAAAATACATTGGAA
GATGTGAAGGTAAAAGAGAAACAGAGTGTTACGCTGACTTGTGAAGTGACAGGGAATC
CAGTGCCAGAAATTACATGGCACAAAGATGGGCAGCCCCTCCAAGAAGATGAAGCCCA

TCACATTATATCTGGTGGCCGTTTTCTTCAAATTACCAATGTCCAGGTGCCACACACT
TGTCACTGTCATCCTTAACAGCCCTACATCTTTGGTCTGTGAAGCTTATTCATATCCT
CCAGCTACCATCACCTGGTTTAAGGATGGCACTCCTTTAGAATCTAACCGAAATATTC
AAGATACTCTTGTGTAGCCACGAATGAGGCTGGAGAAATGATAAAGCACTATGAAGTG
ATGATCATGTTAATATTGCTGCGAATGGACACACACTTCAAATAAAGGAGGCTCAAAT
ATCAGACACCGGACGATATACTTGTGTAGCATCTAACATTGCAGGTGAAGATGAGTTG
GATTTTGATGTGAATATTCAAGTTCCTCCAAGTTTTCAGAAACTCTGGGAAATAGGAA
ACATGCTAGATACTGGCAGGAATGGTGAAGCCAAAGATGTGATCATCAACAATCCCAT
TTCTCTTTACTGTGAGACAAATGCTGCTCCCCCTCCTACACTGACATGGTACAAAGAT
GGCCACCCTCTGACCTCAAGTGATAAAGTATTGATTTTGCCAGGAGGGCGAGTGTTGC
AGATTCCTCGGGCTAAAGTAGAAGATGCTGGGAGATACACATGTGTGGCTGTGAATGA
GGCTGGAGAAGATTCCCTTCAATATGATGTCCGTGTACTCGTGCCGCCAATTATCAAG
GGAGCAAATAGTGATCTCCCTGAAGAGGTCACCGTGCTGGTGAACAAGAGTGCACTGA
TAGAGTGTTTATCCAGTGGCAGCCCAGCACCAAGGAATTCCTGGCAGAAAGATGGACA
GCCCTTGCTAGAAGATGACCATCATAAATTTCTATCTAATGGACGAATTCTGCAGATT
CTGAATACTCAAATAACAGATATCGGCAGGTATGTGTGTGTTGCTGAGAACACAGCTG
GGAGTGCCA~AAATATTTTAACCTCAP.TGTTCATGTTCCTCCAAGTGTCATTGGTCC
TAAATCTGAAAATCTTACCGTCGTGGTGAACAATTTCATCTCTTTGACCTGTGAGGTC
TCTGGTTTTCCACCTCCTGACCTCAGCTGGCTCAAGAATGAACAGCCCATCAAACTGA
TGTGCCTGGTGGTCGAACTCTACAGATTATTCGGGCCAAGGT
TACACTTGTATAGCTATCAATCAAGCTGGCGAAAGCAAGAAA
~vU'r'r't°rcc:c'rGAC'rG r t"1'ATGTGCCCCCAAGCATTAAAGACCATGACAGTGAATCTC
TTTCTGTAGTTAATGTAAGAGAGGGAACTTCTGTGTCTTTGGAGTGTGAGTCGAACGC
TGTGCCACCTCCAGTCATCACTTGGTATAAGAATGGGCGGATGATAACAGAGTCTACT
CATGTGGAGATTTTAGCTGATGGACAAATGCTACACATTAAGAAAGCTGAGGTATCTG
ACACAGGCCAGTATGTATGTAGAGCTATAAATGTAGCAGGACGGGATGATAAAAATTT
CCACCTCAATGTATATGTGCCACCCAGTATTGAAGGACCTGAAAGAGAAGTGATTGTG
GAGACGATCAGCAATCCTGTGACATTAACATGTGATGCCACTGGGATCCCACCTCCCA
CGATAGCATGGTTAAAGAACCACAAGCGCATAGAAAATTCTGACTCACTGGAAGTTCG
TATTTTGTCTGGAGGTAGCAAACTCCAGATTGCCCGGTCTCAGCATTCAGATAGTGGA
AACTATACATGTATTGCTTCAAATATGGAGGGAAAAGCCCAGAAATATTACTTTCTTT
CAATTCAAGTTCCTCCAAGTGTTGCTGGTGCTGAAATTCCAAGTGATGTCAGTGTCCT
TCTAGGAGAAAATGTTGAGCTGGTCTGCAATGCAAATGGCATTCCTACTCCACTTATT
CAATGGCTTAAAGATGGAAAGCCCATAGCTAGTGGTGAAACAGAAAGAATCCGAGTGA
GTGCAAATGGCAGCACATTAAACATTTATGGAGCTCTTACATCTGACACGGGGAAATA
CACATGTGTTGCTACTAATCCCGCTGGAGAAGAAGACCGAATTTTTAACTTGAATGTC
TATG T TACACC TACAATTAGG;GGTAA~T~AAAUAuGAA GCAGAGAAACTAATGACTTTAC.
TGGATACTTCAATAAATATTGAATGCAGAGCCACAGGGACGCCTCCACCACAGATAAA
~CTGGCTGAAGAATGGACTTCCTCTGCCTCTCTCCTCCCATATCCGGTTACTGGCAGCA
~GGACAAGTTATCAGGATTGTGAGAGCTCAGGTGTCTGATGTCGCTGTGTATACTTGTG
'TGGCCTCCAACAGAGCTGGGGTGGATAATAAGCATTACAATCTTCAAGTGTTTGCACC
ACCAAATATGGACAATTCAATGGGGACAGAGGAAATCACAGTTCTCAAAGGTAGTTCC
ACCTCTATGGCATGCATTACTGATGGAACCCCAGCTCCCAGTATGGCCTGGCTTAGAG
ATGGCCAGCCTCTGGGGCTTGATGCCCATCTGACAGTCAGCACCCATGGAATGGTCCT
GCAGCTCCTCAAAGCAGAGACTGAAGATTCGGGAAAGTACACCTGCATTGCCTCAAAT
GAAGCTGGAGAAGTCAGCAAGCACTTTATCCTCAAGGTCCTAGAACCACCTCACATTA
ATGGATCTGAAGAACATGAAGAGATATCAGTAATTGTTAATAACCCACTTGAACTTAC
CTGCATTGCTTCTGGAATCCCAGCCCCTAAAATGACCTGGATGAAAGATGGCCGGCCC
CTTCCACAGACGGATCAAGTGCAAACTCTAGGAGGAGGAGAGGTTCTTCGAATTTCTA
CTGCTCAGGTGGAGGATACAGGAAGATATACATGTCTGGCATCCAGTCCTGCAGGAGA
TGATGATAAGGAATATCTAGTGAGAGTGCATGTACCTCCTAATATTGCTGGAACTGAT
GAGCCCCGGGATATCACTGTGTTACGGAACAGACAAGTGACATTGGAATGCAAGTCAG
ATGCAGTGCCCCCACCTGTAATTACTTGGCTCAGAAATGGAGAACGGTTACAGGCAAC
ACCTCGAGTGCGAATCCTATCTGGAGGGAGATACTTGCAAATCAACAATGCTGACCTA
GGTGATACAGCCAATTATACCTGTGTTGCCAGCAACATTGCAGGAAAGACTACAAGAG
AATTTATTCTCACTGTAAATGTTCCTCCAAACATAAAGGGGGGCCCCCAGAGCCTTGT
AATTCTTTTAAATAAGTCAACTGTATTGGAATGCATCGCTGAAGGTGTCCCAACTCCA

TCTTGGAAAATGGATTCCTTCATATTCAATCAGCACATGTCACTGACACTGGACGGTA
TTTGTGTATGGCCACCAATGCTGCTGGAACAGATCGCAGGCGAATAGATTTACAGGTC
CATGTTCCTCCATCTATTGCTCCGGGTCCTACCAACATGACTGTAATAGTAAATGTTC
AAACTACTCTGGCTTGTGAGGCTACTGGGATACCAAAACCATCAATCAATTGGAGAAA
TTATTTCCCCTTCTGTGGATGACACTGCAACCTATGAATGTACTG
TGACAAACGGTGCTGGAGATGATAAAAGAACTGTGGATCTCACTGTCCAAGTTCCACC
TTCCATAGCTGATGAGCCTACAGATTTCCTAGTAACCAAACATGCCCCAGCAGTAATT
TGGTATAA
.TTCTGTCCTCAGGAGCAATTGAAATACT
TGCCACCCAATTAAACCATGCTGGAAGATACACTTGTGTCGCTAGGAATGCGGCTGGC
CAAGTGAACTACACGTCATTCTGAACAATCCTATTTTATTACCATGTGAAGCAACAGG
GACACCCAGTCCTTTCATTACTTGGCAAAAAGAAGGCATCAATGTTAACACTTCAGGC
CAAGTTAAATGTCCAAGTTCCTCCAGTCATTAGCCCTCATCTAAAGGAATATGTTATT
TTACATGGCATAAAGATGGGCGTGCAATTGTGGAATCTATCCGCCAGCGCGTCCTCAG
CTCTGGCTCTCTGCAAATAACATTTGTCCAGCCTGGTGAfiGCTGGCCATTACACGTGC
CACCCAGGATCAGAAGTACAGAAGGACACTACACGGTCAATGAGAATTCACAAGCCAT
TCTTCCATGCGTAGCTGATGGAATCCCCACACCAGCAATTAACTGGAAAAAAGACAAT
GTTCTTTTAGCTAACTTGTTAGGAAAATACACTGCTGAACCATATGGAGAACTCATTT
TAGAAAATGTTGTGCTGGAGGATTCTGGCTTCTATACCTGTGTTGCTAACAATGCTGC
AGGTGAAGATACACACACTGTCAGCCTGACTGTGCATGTTCTCCCCACTTTTACTGAA
CTTCCTGGAGACGTGTCATTAAATAAAGGAGAACAGCTACGATTAAGCTGTAAAGCTA
CTGGTATTCCATTGCCCAAATTAACATGGACCTTCAATAACAATATTATTCCAGCCCA
CTTTGACAGTGTGAATGGACACAGTGAACTTGTTATTGAAAGAGTGTCAAAAGAGGAT
TCAGGTACTTATGTGTGCACCGCAGAGAACAGCGTTGGCTTTGTGAAGGCAATTGGAT
TTGTGTATGTGAAAGAACCTCCAGTCTTCAAAGGTGATTATCCTTCTCACTGGATTGA
ACCACTTGGTGGGAATGCAATCCTGAATTGTGAGGTGAAAGGAGACCCCACCCCAACC
ATCCAGTGGAACAGAAAGGGAGTGGATATTGAAATTAGCCACAGAATCCGGCAACTGG
GCAATGGCTCCCTGGCCATCTATGGCACTGTTAATGAAGATGCCGGTGACTATACATG
TGTAGCTACCAATGAAGCTGGGGTGGTGGAGCGCAGCATGAGTCTGACTCTGCAAAGT
CCTCCTATTATCACTCTTGAGCCAGTGGAAACTGTTATTAATGCTGGTGGCAAAATCA
TATTGAATTGTCAGGCAACTGGAGAGCCTCAACCAACCATTACATGGTCCCGTCAAGG
GCACTCTATTTCCTGGGATGACCGGGTTAACGTGTTGTCCAACAACTCATTATATATT
GCTGATGCTCAGAAAGAAGATACCTCTGAATTTGAATGTGTTGCTCGAAACTTAATGG
GTTCTGTCCTTGTCAGAG T GCCAGVTCATAG'T'CCAGGTTCATt;C_aTGGATTTTCrCprT,_;
GTCTGCATGGAGAGCCTGCAGTGTCACCTGTGGAAAAGGCATCCAAAAGAGGAGTCGT
CTGTGCAACCAGCCCCTTCCAGCCAATGGTGGGAAGCCCTGCCAAGGTTCAGATTTGG
TCTTTGGGAAGAATGCACAAGGAGCTGTGGACGCGGCAACCAAACCAGGACCAGGACT
TCCATCAGTTCAGCATGGTGGGCGGCCATGTGAAGGGAATGCTGTGGAAA
AATAACCCACCACCAGCGTTTGGTGGGTCCTACTGTGATGGAGCAGAAACACAGATGC
AAGTTTGCAATGAAAGAAATTGTCCAGTTCATGGCAAGTGGGCGACTTGGGCCAGTTG
GAGTGCCTGTTCTGTGTCATGTGGAGGAGGTGCCAGACAGAGAACAAGGGGCTGCTCC
GACCCTGTGCCCCAGTATGGAGGAAGGAAATGCGAAGGGAGTGATGTCCAGAGTGATT
TTTGCAACAGTGACCCTTGCCCAACCCATGGTAACTGGAGTCCTTGGAGTGGCTGGGG
AACATGCAGCCGGACGTGTAACGGAGGGCAGATGCGGCGGTACCGCACATGTGATAAC
CCTCCTCCCTCCAATGGGGGAAGAGCTTGTGGGGGACCAGACTCCCAGATCCAGAGGT
GCAACACTGACATGTGTCCTGTGGATGGAAGTTGGGGAAGCTGGCATAGTTGGAGCCA
GTGCTCTGCCTCCTGTGGAGGAGGTGAAAAGACTCGGAAGCGGCTGTGCGACCATCCT
GTGCCAGTTAAAGGTGGCCGTCCCTGTCCCGGAGACACTACTCAGGTGACCAGGTGCA
ATGTACAAGCATGTCCAGGTGGGCCCCAGCGAGCCAGAGGAAGTGTTATTGGAAATAT
TAATGATGTTGAATTTGGAATTGCTTTCCTTAATGCCACAATAACTGATAGCCCTAAC
TCTGATACTAGAATAATACGTGCCAAAATTACCAATGTACCTCGTAGTCTTGGTTCAG

CAATGAGAAAGATAGTTTCTATTCTAAATCCCATTTATTGGACAACAGCAAAGGAAAT

AGGAGAAGCAGTCAATGGCTTTACCCTCACCAATGCAGTCTTCAAAAGAGAAACY'CAA

GTGGAATTTGCAACTGGAGAAATCTTGCAGATGAGTCATATTGCCCGGGGCTTGGATT

CCGATGGTTCTTTGCTGCTAGATATCGTTGTGAGTGGCTATGTCCTACAGCTTCAGTC

ACCTGCTGAAGTCACTGTAAAGGATTACACAGAGGACTACATTCAAACAGGTCCTGGG

CAGCTGTACGCCTACTCAACCCGGCTGTTCACCATTGATGGCATCAGCATCCCATACA

CATGGAACCACACCGTTTTCTATGATCAGGCACAGGGAAGAATGCCTTTCTTGGTTGA

AACACTTCATGCATCCTCTGTGGAATCTGACTATAACCAGATAGAAGAGACACTGGGT

TTTAAAATTCATGCTTCAATATCCAAAGGAGATCGCAGTAATCAGTGCCCCTCCGGGT

TTACCTTAGACTCAGTTGGACCTTTTTGTGCTGATGAGGATGAATGTGCAGCAGGGAA

TCCCTGCTCCCATAGCTGCCACAATGCCATGGGGACTTACTACTGCTCCTGCCCTAAA

GGCCTCACCATAGCTGCAGATGGAAGAACTTGTCAAGATATTGATGAGTGTGCTTTGG

GTAGGCATACCTGCCACGCTGGTCAGGACTGTGACAATACGATTGGATCTTATCGCTG

TGTGGTCCGTTGTGGAAGTGGCTTTCGAAGAACCTCTGATGGGCTGAGTTGTCAAGAT

ATTAATGAATGTCAAGAATCCAGCCCCTGTCACCAGCGCTGTTTCAATGCCATAGGAA

GTTTCCATTGTGGATGTGAACCTGGGTATCAGCTCAAAGGCAGAAAATGCATGGATGT

GAACGAGTGTAGACAAAATGTATGCAGACCAGATCAGCACTGTAAGAACACCCGTGGT

GGCTATAAGTGCATTGATCTTTGTCCAAATGGAATGACCAAGGCAGAAAATGGAACCT

GTATTGATATTGATGAATGTAAAGATGGGACCCATCAGTGCAGATATAACCAGATATG

TGAGAATACAAGAGGCAGCTATCGTTGTGTATGCCCAAGAGGTTATCGGTCTCAAGGA

GTTGGAAGACCCTGCATGGACATTAATGAATGTGP.A.CAAGTGCCTan,~CCT_rGmr_raC

ATCAGTGCTCCAACACCCCCGGCAGCTTCAAGTGTATCTGTCCACCAGGACAACATTT

ATTAGGGGACGGGAAATCTTGCGCTGGATTGGAGAGGCTGCCAAATTATGGCACTCAA

TACAGTAGCTATAACCTTGCACGGTTCTCCCCTGTGAGAAACAACTATCAACCTCAAC

AGCATTACAGACAGTACTCACATCTCTACAGCTCCTACTCAGAGTATAGAAACAGCAG

AACATCTCTCTCCAGGACTAGAAGGACTATTAGGAAAACTTGCCCTGAAGGCTCTGAG

GCAAGCCATGACACATGTGTAGATATTGATGAATGTGAAAATACAGATGCCTGCCAGC

ATGAGTGTAAGAATACCTTTGGAAGTTATCAGTGCATCTGCCCACCTGGCTATCAACT

CACACACAATGGAAAGACATGCCAAGATATCGATGAATGTCTGGAGCAGAATGTGCAC

TGTGGACCCAATCGCATGTGCTTCAACATGAGAGGAAGCTACCAGTGCATCGATACAC

CCTGTCCACCCAACTACCAACGGGATCCTGTTTCAGGGTTCTGCCTCAAGAACTGTCC

ACCCAATGATTTGGAATGTGCCTTGAGCCCATATGCCTTGGAATACAAACTCGTCTCC

CTCCCATTTGGAATAGCCACCAATCAAGATTTAATCCGGCTGGTTGCATACACACAGG

ATGGAGTGATGCATCCCAGGACAACTTTCCTCATGGTAGATGAGGAACAGACTGTTCC

TTTTGCCTTGAGGGATGAAAACCTGAAAGGAGTGGTGTATACAACACGACCACTACGA

GAAGCAGAGACCTACCGCATGAGGGTCCGAGCCTCATCCTACAGTGCCAATGGGACCA

TTGAATATCAGACCACATTCATAGTTTATATAGCTGTGTCCGCCTATCCATACTAA
GG

_ AACTCTCCAAAGCCTATTCCACATATTTAAACCGCATTAATCATGGCAATCAAGCCCC

CTTCCAGATTACTGTCTCTTGAACAGTTGCAATCTTGGCAGCTTGAAAATGGTGCTAC

ACTCTGTTTTGTGTGCCTTCCTTGGTACTTCTGAGGTATTTTCATGATCCCACCATGG

TCATATCTTGAAGTATGGTCTAGAAAAGTCCCTTATTATTTTATTTATTAC_nCTGGAr CAGTTACTTCCCAAAGATTATTCTGAACATCTAACAGGACATATCAGTGATGGTTTAC

AGTAGTGTAGTACCTAAGATCATTTTCCTGAAAGCCAAACCAAACAACGAAAAACAAG

AACAACTAATTCAGAATCAAATAGAGTTTTTGAGCATTTGACTATTTTTAGAATCATA

AAATTAGTTACTAAGTATTTTGATCAAAGCTTATAAAATAACTTACGGAGATTTTTGT

AAGTATTGATACATTATAATAGGACTTGCCTATTTTCATTTTTAAGAAGAAAAACCCG

ORF Start: ATG at 1649 ORF Stop:
TAA at 15134 SEQ ID NO: 20 4495 as MW at 488830.SkD

NOV6b, MWLDRCARNPPRNHIDAPQKAPSAFRRVLQKGELISTSSAKFSAGADGSLYWSPGGE

Protein Sequence~SLPPPIITWAKETQLISPFSPRHTFLPSGSMKiTETRTSDSGMYLCVATNIAGNVT

QAVKLNVHVPPKIQRGPKHLKVQVGQRVDIPCNAQGTPLPVITWSKGGSTMLVDGEHH

VSNPDGTLSIDQATPSDAGIYTCVATNIAGTDETEITLHVQEPPTVEDLEPPYNTTFQ

ERVANQRIEFPCPAKGTPKPTIKWLFINGRELTGREPGISILEDGTLLVIASVTPYDNG

EYICVAVNEAGTTERKYNLKVH~7PpVIKDKEQVTNVSVLLNQLTNLFCEVEGTPSPII

MWYKDNVQVTESSTIQTVNNGKILKLFRATPEDAGRYSCKAINIAGTSQKYFNIDVLG

TNFPNEVSWLN'RDVALECQVKGTPFPDIHWFKDGNIKGGNVTTDISVLINSLIKLEC

ETRGLPMPAITWYKDGQPIMSSSQALYIDKGQYLHIPRAQVSDSATYTCHVANVAGTA

EKSFHVDVYVPPMIEGNLATPLNKQWIAHSLTLECKAAGNPSPILTWLKDGVPVKAN

FIVMVNNLLELDCHVTGSPPPTIMWLKDGQLIDERDGFKILLNGRKLVIAQAQVSNTG
LYRCMAANTAGDHKKEFEVTVHVPPTIKSSGLSERVVVICYKPVALQCIANGIPNPSIT
WLKDDQPVNTAQGNLKIQSSGRVLQIAKTLLEDAGRYTCVATNAAGETQQHIQLHVHE
PPSLEDAGKMLNETVLVSNPVQLECKAAGNPVPVITWYKDNRLLSGSTSMTFLNRGQI
IDIESAQISDAGIYKCVAINSAGATELFYSLQVHVAPSISGSNNMVAVVVNNPVRLEC
EARGIPAPSLTWLKDGSPVSSFSNGLQVLSGGRILALTSAQISDTGRYTCVAVNAAGE
KQRDIDLRVYVPPNIMGEEQNVSVLISQAVELLCQSDAIPPPTLTWLKDGHPLLKKPG
LSISENRSVLKIEDAQVQDTGRYTCEATNVAGKTEXZJYNVNIWVPPNIGGSDELTQLT
VIEGNLISLLCESSGIPPPNLIWKKKGSPVLTDSMGRVRILSGGRQLQISIAEKSDAA
LYSCVASNVAGTAKKEYNLQVYIRPTITNSGSHPTEIIVTRGKSISLECEVQGIPPPT
VTWMKDGHPLIKAKGVEILDEGHILQLKNIHVSDTGRYVCVAVNVAGMTDKKYDLSVH
GGRMLRLMQTTMEDAGQYTCWRNAAGEERKIFGLSVLVPPHIVGENTLEDVKVKEKQ
SVTLTCEVTGNPVPEITWHKDGQPLQEDEAHHIISGGRFLQITNVQVPHTGRYTCLAS
SPAGHKSRSFSLNVFVSPTIAGVGSDGNPEDVTVILNSPTSLVCEAYSYPPATITWFK
DGTPLESNRNIRILPGGRTLQILNAQEDNAGRYSCVATNEAGEMIKHYEVKVYTLNAN
IVIIESQPLKSDDHVNIAANGHTLQIKEAQISDTGRYTCVASNIAGEDELDFDVNIQV
PPSFQKLWEIGNMLDTGRNGEAKDVIINNPISLYCETNAAPPPTLTWYKDGHPLTSSD
KVLILPGGRVLQIPRAKVEDAGRYTCVAVNEAGEDSLQYDVRVLVPPIIKGANSDLPE
EVTVLVNKSALIECLSSGSPAPRNSWQKDGQPLLEDDHHKFLSNGRILQILNTQTTDI
GRYVCVAENTAGSAKKYFNLNVHVPPSVIGPKS~~TV~7Vr'!NFT_ ST_.m~crFPPPZ,T.
SWLKNEQPIKLNTNTLIVPGGRTLQIIRAKVSDGGEYTCIAINQAGESKKKFSLTVYV
PPSIKDHDSESLSVVNVREGTSVSLECESNAVPPPVITWYKNGRMITESTHVEILADG
QMLHIKKAEVSDTGQYVCRAINVAGRDDKNFHLNVYVPPSIEGPEREVIVETISNPVT
'LTCDATGIPPPTIAWLKNHKRIENSDSLEVRILSGGSKLQIARSQHSDSGNYTCIASN
MEGKAQKYYFLSIQVPPSVAGAEIPSDVSVLLGENVELVCNANGIPTPLIQWLKDGKP
',IASGETERIRVSANGSTLNIYGALTSDTGKYTCVATNPAGEEDRIFNLNVYVTPTIRG
NKDEAEKLMTLVDTSINIECRATGTPPPQINWLKNGLPLPLSSHTRLLAAGQVIRIVR
AQVSDVAVYTCVASNRAGVDNKHYNLQVFAPPNMDNSMGTEEITVLKGSSTSMACITD
GTPAPSMAWLRDGQPLGLDAHLTVSTHGMVLQLLKAETEDSGKYTCIASNEAGEVSKH
FILKVLEPPHINGSEEHEEISVIVNNPLELTCIASGIPAPKMTWMKDGRPLPQTDQVQ
TLGGGEVLRISTAQVEDTGRYTCLASSPAGDDDKEYLVRVHVPPNIAGTDEPRDITVL
RNRQVTLECKSDAVPPPVITWLRNGERLQATPRVRILSGGRYLQINNADLGDTANYTC
VASNIAGKTTREFILTVNVPPNIKGGPQSLVILLNKSTVLECIAEGVPTPRITWRKDG
AVLAGNHARYSILENGFLHIQSAHVTDTGRYLCMATNAAGTDRRRIDLQVHVPPSIAP
GPTNMTVIVNVQTTLACEATGIPKPSINWRKNGHLLNVDQNQNSYRLLSSGSLVIISP
SVDDTATYECTVTNGAGDDKRTVDLTVQVPPSIADEPTDFLVTKHAPAVITCTASGVP
FPSIHWTKNGIRLLPRGDGYRILSSGAIEILATQLNHAGRYTCVARNAAGSAHRHVTL
HVHEPPVIQPQPSELHVILNNPILLPCEATGTPSPFITWQKEGINVNTSGRNHAVLPS
GGLQISRAVREDAGTYMCVAQNPAGTALGKIKLNVQVPPVISPHLKEYVIAVDKPITL, ~CEADGT PPPDITWHk77GRAZVESIRQR'v'LSSGSLQIT~'T~QPGDAGHYTCMA.~TNVAGS
SSTSTKLTVHVPPRIRSTEGHYTVNENSQAILPCVADGIPTPATNWKKDNVLLANLLG
KYTAEPYGELILENVVLEDSGFYTCVANNAAGEDTHTVSLTVHVLPTFTELPGDVSLN
KGEQLRLSCKATGIPLPKLTWTFNNNIIPAHFDSVNGHSELVIERVSKEDSGTYVCTA
ENSVGFVKAIGFVYVKEPPVFKGDYPSHWIEPLGGNAILNCEVKGDPTPTIQWNRKGV
DIEISHRIRQLGNGSLAIYGTVNEDAGDYTCVATNEAGVVERSMSLTLQSPPIITLEP
VETVINAGGKIILNCQATGEPQPTITWSRQGHSISWDDRVNVLSNNSLYIADAQKEDT
SEFECVARNLMGSVLVRVPVIVQVHGGFSQWSAWRACSVTCGKGIQKRSRLCNQPLPA
MRNCQNKPCPVDGSWSEWSLWEECTRSCGRGNQTRTRTCNNPSVQH
IMCNIRPCPVHGAWSAWQPWGTCSESCGKGTQTRARLCNNPPPAFG
RKCEGSDVQSDFCNSDPCPTHGNWSPWSGWGTCSRTCNGGQMRRYRTCDNPPPSNGGR
ACGGPDSQIQRCNTDMCPVDGSWGSWHSWSQCSASCGGGEKTRKRLCDHPVPVKGGRP
CPGDTTQVTRCNVQACPGGPQRARGSVIGNINDVEFGIAFLNATITDSPNSDTRIIRA
KITNVPRSLGSAMRKIVSILNPIYWTTAKEIGEAVNGFTLTNAVFKRETQVEFATGEI
LQMSHIARGLDSDGSLLLDIVVSGYVLQLQSPAEVTVKDYTEDYIQTGPGQLYAYSTR
LFTIDGISIPYTWNHTVFYDQAQGRMPFLVETLHASSVESDYNQIEETLGFKIHASIS
KGDRSNQCPSGFTLDSVGPFCADEDECAAGNPCSHSCHNAMGTYYCSCPKGLTIAADG
RTCQDIDECALGRHTCHAGQDCDNTIGSYRCVVRCGSGFRRTSDGLSCQDINECQESS
PCHQRCFNAIGSFHCGCEPGYQLKGRKCMDVNECRQNVCRPDQHCKNTRGGYKCIDLC
PNGMTKAENGTCIDIDECKDGTHQCRYNQICENTRGSYRCVCPRGYRSQGVGRPCMDI

NECEQVPKPCAHQCSNTPGSFKCICPPGQHLLGDGKSCAGLERLPNYGTQYSSYNLAR
FSPVRNNYQPQQHYRQYSHLYSSYSEYRNSRTSLSRTRRTIRKTCPEGSEASHDTCVD
IDECENTDACQHECKNTFGSYQCICPPGYQLTHNGKTCQDIDECLEQNVHCGPNRMCF
QDLIRLVAYTQDGVNgiPRTTFLMVDEEQTVPFALRDENLKGVVYTTRPLREAETYRM12 VRASSYSANGTIEYQTTFIVYIAVSAYPY
Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 6B.
Table 6B. Comparison of NOV6a against NOV6b.
Protein Sequence NOV6a Residues/ Identities/
Match Residues Similarities for the Matched Region NOV6b 1..707 698/707 (98%) 2917..3623 701/707 (98%) Further analysis of the NOV6a protein yielded the following properties shown in Table 6C.
Table 6C. Protein Sequence Properties NOV6a PSort 0.4500 probability located in cytoplasm; 0.3000 probability located in microbody analysis: (peroxisome); 0.1000 probability located in mitochondrial matrix space; 0.1000 probability located in lysosome (lumen) SignalP No Known Signal Sequence Predicted analysis: .
~A search of the NOV6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 6D.
Table 6D. Geneseq Results for NOV6a NOV6a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegian AAB4777IHuman thrombospondin protein,1..707 699/707 (98%)0.0 BTL.012 - Homo sapiens, 184..890702/707 (98%) 1336 aa.

[WO200174852-A2, 11-OCT-2001]

ABG03933Novel human diagnostic protein1..471 471/471 (100%)0.0 #3924 -Homo sapiens, 1240 aa. 442..912471/471 (100%) [W0200175067-A2, I I-OCT-2001]

ABG03933 Novel human diagnostic protein1..471 471/471 (100%) 0.0 #3924 -Homo sapiens, 1240 aa. 442..912471/471 (100%) [W0200175067-A2, I I-OCT-2001]

AAG67244 Amino acid sequence of murine395..707246/313 (78%) e-164 thrombospondin 1-like protein - Mus 1..313 280/313 (88%) musculus, 1068 aa. [W0200109321-Al, AAB47770 Human thrombospondin protein,471..678208/208 (100%) e-135 BTL.012, fragment 654-861 -Homo 1..208 208/208 (100%) Sapiens, 208 aa. [W0200174852-A2, In a BLAST search of public sequence datbases, the NOV6a protein was found to have homology to the proteins shown in the BLASTP data in Table 6E.
Table 6E. Public BLASTP Results for NOV6a NOV6a Identities/

Protein Residues/SimilaritiesExpect for AccessionProtein/Organism/Length Match the MatchedValue Number ResiduesPortion 1 Q96RW7 HEMICENTIN - Homo Sapiens 1..707 700/707 ~ 0.0 (Human), (99%) 5636 aa. 4058..4764701/707 (99%) Q96SC3 FIBULIN-6 - Homo sapiens (Human),1..707 698/707 0.0 2673 (98%) as (fragment). 1095..1801701/707 ~
(98%) Q96DN3 CDNA FLJ31995 FIS, CLONE 1..460 159/475 8e-64 (33%) NT2RP7009236, WEAKLY SIMILAR 782..12522351475 ~
TO (49%) BASEMENT MEMBRANE-SPECIFIC

HEPARAN SULFATE PROTEOGLYCAN

CORE PROTEIN PRECURSOR - Homo Sapiens (Human), 1252 as (fragment):

T20992 hypothetical protein F15G9.4a-2..511 159/529 1e-59 ~ (30%) Caenorhabditis elegans, 5175 3014..3521241/529 aa. (45%) 076518 HEMICENTIN PRECURSOR- 2..511 159/529 1e-59 (30%) ~ ~ ~
Caenorhabditis elegans, S 3014..3521241 /529 198 aa. (45%) PFam analysis predicts that the NOV6a protein contains the domains shown in the Table 6F.

Table 6F. Domain Analysis of NOV6a Identities/

Pfam DomainNOV6a Match Similarities Expect Region Value for the Matched Region Ig 37..94 19161 (31 %) 1.1 e-10 43161 (70%) Ig 127..185 16/62 (26%) 1 e-08 39/62 (63%) Ig 218..274 20/60 (33%) 9.5e-12 43/60 (72%) Ig 308..365 20/61 (33%) 2.7e-10 42/6 i (69%) Ig 398..455 17/61 (28%) ~ 1.6e-09 42/61 (69%) tsp 1 477..527 28/54 (52%) 1.1 e-16 3 69%) ( 7/54 tsp 1 534..584 25/54 (46%) 5.7e-14 4 I /54 (76%) tsp 1 591..641 22/54 (41 %) 4e-12 3 67%) ( 6154 tsp 1 648..698 23/54 (43%) 1.9e-14 ( 69%) Example 7.
The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 7A.

TGGACGCTTCCCGGGCCCAGTTCTTCCTGCACTTGTCCCCAAGCCACTATGCTCTGCTI
TGCAGAAAAGTAGCAGTTCTGAAGATGGGTCAATGGGGAGCTTTTCGGAGAAGTCTAGI
CTCGGCCTGAGCGGCTGTATATCCAGGTGTTCTTGAAGAAGGATGACTCAGTGGGCTAI
CCGGGCTTTGGTGCAGACAGAGGATCATCTGCTACTTTTCCTGCAGCAGTTGGGGAAG
GTGGTGCTGTGGAGCCGTGAGGAGTCCCTGGCAGAAGTGGTGTGCCTAGAGATGGTGG
TTTGGCAAAAAGGCAGAI
TGGCTTGCTGGGGATGTTCCTGAAACGCCTCTCGTCTCAGCTTATCCTGCTGCAAGCAi GGTGATGGTAACAGCCTCAGGCAAGCTTTTTGGCATTGAGAGCAGCTCTGGCACCATC
CTGTGGAAACAGTATCTACCCAATGTCAAGCCAGACTCCTCCTTTAAACTGATGGTCCI
GTAGCTCCCCCAGTGCTGAAGCGCCCCATCTTGCAGTCCTTGCTTCTCCCAGTCATGG
ATCAAGACTACGCCAAGGTGTTGCTGTTGATAGATGATGAATACAAGGTCACAGCTTT
TCCAGCCACTCGGAATGTCTTGCGACAGCTACATGAGCTTGCCCCTTCCATCTTCTTC
TATTTGGTGGATGCAGAGCAGGGACGGCTGTGTGGATATCGGCTTCGAAAGGATCTCA
CCACTGAGCTGAGTTGGGAGCTGACCATTCCCCCAGAAGTACAGCGGATCGTCAAGGT
GAAGGGGAAACGCAGCAGTGAGCACGTTCATTCCCAGGGCCGTGTGATGGGGGACCGC
TATCATTCACTCCTCTGTGCAGAAGAAAGCCAAAGGCCCTGTCCATATCGTGCATTCAI
GAGAACTGGGTGGTGTACCAGTACTGGAACACCAAGGCTCGGCGCAACGAGTTTACCG
GCCATGGAGGCCACCATCACCGAACGGGGCATCACCAGCCGACACCTGCTGATTGGACI
ORF Start: ATG at 21 ORF Stop: TAA at 2997 SEQ ID NO: 22 992 as MW at 1116S9.1kD
NOV7a, MAAEWASRFWLWATLLIPAAAVYEDQVGKFDWRQQYVGKVKFASLEFSPGSKKLVVAT

Protein Sequence ~EITLDSGSFQALGLVGLQESVRYIAVLKKTTLALHIiLSSGHLKWVEHLPESDSIH
YQMVYSYGSGVVWALGVVPFSHVNIVKFNVEDGEIVQQVKVSTPWLQHLSGACGVVDE
AVLVCPDPSSRSLQTLALETEWELRQIPLQSLDLEFGSGFQPRVLPTQPNPVDASRAQ
FFLHLSPSHYALLQYHYGTLSLLKNFPQTALVSFATTGEKTVAAVMACRNEVQKSSSS
EDGSMGSFSEKSSSKDSLACFNQTYTINLYLVETGRRLLDTTITFSLEQSGTRPERLY
QAELEGEFGKKADGLLGMFLKRLSSQLILLQAWTSHLWKMFYDARKPRSQIKNEINID' TLARDEFNLQRN~IVMVTASGKLFGIESSSGTILWKQYLPNVKPDSSFKLMVQRTTAHF
PHPPQCTLLVKDKESGMSSLYVFNPIFGKWSQVAPPVLKRPILQSLLLPVMDQDYAKV'i LLLIDDEYKVTAFPATRNVLRQLHELAPSIFFYLVDAEQGRLCGYRLRKDLTTELSWEj IIO

FIGIFLIDGVTGRIIIiSSVQKKAKGPVHIVHSENWWYQYWNTKARRNEFTVLELYEG
TEQYNATAFSSLDRPQLPQVLQQSYIFPSSISAMEATITERGITSRHLLIGLPSGAIL
SLPKALLDPRRPEIPTEQSREENLIPYSPDVQIHAERFINYNQTVSRMRGIYTAPSGL
ESTCLVVAYGLDIYQTRVYPSICQFDVLKDDYDYVLISSVLFGLVFATMITKRLAQVKL
LNR.AWR
Further analysis of the NOV7a protein yielded the following properties shown in Table 7B.
Table 7B. Protein Sequence Properties NOV7a PSort 0.4600 probability located in plasma membrane; 0.2800 probability located in analysis: endoplasmic reticulum (membrane); 0.2000 probability located in lysosome (membrane); 0.1875 probability located in microbody (peroxisome) SignalP Cleavage site between residues 22 and 23 analysis:
A search of the NOV7a protein against the Geneseq database, a proprieta:y datr.base u'~at contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7C.
Table 7C. Geneseq Results for NOV7a NOV7a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for Identifier Date] Match the Matched Value ResiduesRegion AAB88468 Human membrane or secretoryI ..992 963/993 (96%)0.0 protein clone PSEC0263 - Homo Sapiens,1..971 967/993 (96%) aa. [EP1067182-A2, 10-JAN-2001) AAE0707S Human gene 2 encoded secreted508..992~ 485/485 ~ 0.0 protein (100%) ~ 1. d85 485/485 ( HPJCP79, SEQ ID NO:92 - 100,! ) Homo sapiens, 485 aa. [W0200I54708-Al, 02- ' AUG-2001 ]

AAE07074 Human gene 2 encoded secreted1..354 352/354 (99%)0.0 protein HPJCP79, SEQ ID N0:91 - 1..354 354/354 (99%) Homo Sapiens, 360 aa. [W0200154708-A1, AUG-2001 ]

ABBS9498 Drosophila melanogaster 440..992252/568 (44%)e-128 polypeptide SEQ ID NO 5286 - Drosophila363..915350/568 (61%) melanogaster, 9I S aa. [W0200171042-A2, 27-SEP-2001 AAY6S107 Human S' EST related polypeptide37..160 114/124 (91%)Se-59 SEQ

ID N0:1268 - Homo Sapiens, 2..125 117/124 (93%) 132 aa.

[W0995305 1-A2, 21-OCT-1999]

In a BLAST search of public sequence datbases, the NOV7a protein was found to have homology to the proteins shown in the BLASTP data in Table 7D.
Table 7D. Public BLASTP Results for NOV7a NOV7a Identities/

Protein Residues/SimilaritiesExpect for AccessionProtein/OrganismlLength Match the Matched Value Number ResiduesPortion CAC39832 SEQUENCE 303 FROM PATENT 1..992 963/993 (96%)0.0 EP1067182 - Homo Sapiens 1..971 967/993 (96%) (Human), 971 aa.

Q14700 KIAA0090 PROTEIN - Homo 89..992 903/905 (99%)0.0 Sapiens (Human), 905 as 1..905 904/905 (99%) (fragment).

Q9NUCI DJ657E11.5 (KIAA0090 PROTEIN)97..992 ' 81J/928 ' 0.C
(87,') ' - Homo Sapiens (Human), 1..847 815/928 (87%) 847 as (fragment).

Q9VHY6 CG2943 PROTEIN - Drosophila440..992252/568 (44%)e-127 melanogaster (Fruit fly), 363..915350/568 (61%) 9I5 aa.

Q95TQ6 LD30573P - Drosophila melanogaster470..992233/531 (43%)e-119 (Fruit fly), 521 aa. 6..521 329/531 (61%) PFam analysis predicts that the NOV7a protein contains the domains shown in the Table 7E.
Table 7E. Domain Analysis of NOV7a Identities/
Pfaln Domain NOV7a Match Region Similarities ~ E.xpeci v dine' for the Matched Region Bacterial_PQQ 52..89 9/38 (24%) 0.19 24/38 (63%) Example 8.
The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.
Table 8A. NOV8 Sequence Analysis ~ SEQ ID NO: 23 ~ 1913 by DNA

Sequence CTGCCCCGGCTGCTGCTGCTGGTGCTGTTGTGCCTGCCGGCCGTGTGGGGTGACTGTG

GCCTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGCCGTACAAGTTTTCC

CGAGGATACTGTAATAACGTACAAATGTGAAGAAAGCTTTGTGAAAATTCCTGGCGAG

AAGGACTCAGTGATCTGCCTTAAGGGCAGTCAATGGTCAGATATTGAAGAGTTCTGCA

ATCGTAGCTGCGAGGTGCCAACAAGGCTAAATTCTGCATCCCTCAAACAGCCTTATAT

CACTCAGAATTATTTTCCAGTCGGTACTGTTGTGGAATATGAGTGCCGTCCAGGTTAC

AGAAGAGAACCTTCTCTATCACCAAAACTAACTTGCCTTCAGAATTTAAAATGGTCCA

CAGCAGTCGAATTTTGTAAAAAGAAATCATGCCATAATCCGGGAGAAATACGAAATGG

TCAGATTGATGTACCAGGTGGCATATTATTTGGTGCAACCATCTCCTTCTCATGTAAC

ACAGGGTACAAATTATTTGGCTCGACTTCTAGTTTTTGTCTTATTTCAGGCAGCTCTG

TCCAGTGGAGTGACCCGTTGCCAGAGTGCAGAGGAAAATCTCTAACTTCCAAGGTCCC

ACCAACAGTTCAGAAACCTACCACAGTAAATGTTCCAAATACAGAATTCTCACCAACT

TCTCAGAAAACCACCACAAAAACCACCACACCAAATGCTCAAGCAACACGGAGTACAC

CCGTTTCCAGGACAACCAAGCATTTTCATGAAACAACCCCAAATAAAGGAAGAGGAAC

CACTTCAGGTACTACCCGTCTTCTATCTGGGCACACGTGTTTCACGTTGACAGGTTTG

CTTGGGACGCTAGTAACCATGGGCTTGCTGACTTAGCCAAAGAAGAGTTAAGAAGAAA

ATACACACAAGTATACAGACTGTTCCTAGTTTCTTAGACTTATCTGCATATTGGATAA

AATAAATGCAATTGTGCTCTTCATTTAGGATGCTTTCATTGTCTTTAAGATGTGTTAG

GAATGTCAACAGAGCAAGGAGAAAAAAGGCAGTCCTGGAATCACATTCTTAGCACACC

TACACCTCTTGAAAATAGAACAACTTGCAGnpmTGaGAGTGAmmCr_mmmCCm~ayz~p,GTI

GTAAGAAAGCATAGAGATTTGTTCGTATTTAGAATGGGATCACGAGGAAAAGAGAAGG

AAAGTGATTTTTTTCCACAAGATCTGTAATGTTATTTCCACTTATAAAGGAAATAAAA

AATGAAAAACATTATTTGGATATCAAAAGCAAATAAAAACCCAATTCAGTCTCTTCTA

AGCAAAATTGCTAAAGAGAGATGAACCACATTATAAAGTAATCTTTGGCTGTAAGGCA

TTTTCATCTTTCCTTCGGGTTGGCAAAATATTTTAAAGGTAAAACATGCTGGTGAACC

AGGGGTGTTGATGGTGATAAGGGAGGAATATAGAATGAAAGACTGAATCTTCCTTTGT

TGCACAAATAGAGTTTGGAAAAAGCCTGTGAAAGGTGTCTTCTTTGACTTAATGTCTT

TAAAAGTATCCAGAGATACTACAATATTAACATAAGAAAAGATTATATATTATTTCTG

AATCGAGATGTCCATAGTCAAATTTGTAAATCTTATTCTTTTGTAATATTTATTTATA

TTTATTTATGACAGTGAACATTCTGATTTTACATGTAAAACAAGAAAAGTTGAAGAAG

ATATGTGAAGAAAAATGTATTTTTCCTAAATAGAAATAAATGATCCCATTTTTTGGT

ORF Start: ATG at 66 ORF Stop: TAG
at 1020 SEQ ID NO: 24 318 as MW at 34479.IkD

NOVBa, MTVARPSVPAALPLLGELPRLLLLVLLCLPAVWGDCGLPPDVPNAQPALEGRTSFPED

CGS7279-O2 '~I~CEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQ

Protein Sequencet'T~FPVGTV~YECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCHIdPGEIRNGQI

DVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRGKSLTSKVPPT

VQKPTTVNVPNTEFSPTSQKTTTKTTTPNAQATRSTPVSRTT__rC~-?FFhTTP_rTICGRGTTS

~
GTTRLLSGh rCFTLTGI~LGTL'iiWiGLL~?~

SEQ ID NO: 2S 1962 by NOV8b, CTGCAAACTTGCATGTCATCTCTTTCAGGTGACTGTGGCCTTCCCCCAGATGTACCTA

DNA

Sequence ATGTGAAGAAAGCTTTGTGAAAATTCCTGGCGAGAAGGACTCAGTGATCTGCCTTAAG

GGCAGTCAATGGTCAGATATTGAAGAGTTCTGCAATCGTAGCTGCGAGGTGCCAACAA

GGCTAAATTCTGCATCCCTCAAACAGCCTTATATCACTCAGAATTATTTTCCAGTCGG

TACTGTTGTGGAATATGAGTGCCGTCCAGGTTACAGAAGAGAACCTTCTCTATCACCA

AAACTAACTTGCCTTCAGAATTTAAAATGGTCCACAGCAGTCGAATTTTGTAAAAAGA

AATCATGCCCTAATCCGGGAGAAATACGAAATGGTCAGATTGATGTACCAGGTGGCAT

ATTATTTGGTGCAACCATCTCCTTCTCATGTAACACAGGGTACAAATTATTTGGCTCG

ACTTCTAGTTTTTGTCTTATTTCAGGCAGCTCTGTCCAGTGGAGTGACCCGTTGCCAG

AGTGCAGAGAAATTTATTGTCCAGCACCACCACAAATTGACAATGGAATAATTCAAGG

GGAACGTGACCATTATGGATATAGACAGTCTGTAACGTATGCATGTAATAAAGGATTC

ACCATGATTGGAGAGCACTCTATTTATTGTACTGTGAATAATGATGAAGGAGAGTGGA

GTGGCCCACCACCTGAATGCAGAGGAAAATCTCTAACTTCCAAGGTCCCACCAACAGT

TCAGAAACCTACCACAGTAAATGTTCCAACTACAGAAGTCTCACCAACTTCTCAGAAA

ACCACCACAAAAACCACCACACCAAATGCTCAAGCAACACGGAGTACACCTGTTTCCA

. GGACAACCAAGCATTTTCATGAAACAACCCCAAATAAAGGAAGTGGAACCACTTCAGG

TACTACCCGTCTTCTATCTGGGCACACGTGTTTCACGTTGACAGGTTTGCTTGGGACG

CTAGTAACCATGGGCTTGCTGACTTAGCCAAAGAAGAGTTAAGAAGAAAATACACACA

AGTATACAGACTGTTCCTAGTTTCTTAGACTTATCTGCATATTGGATAAAATAAATGC

AATTGTGCTCTTCATTTAGGATGCTTTCATTGTCTTTAAGATGTGTTAGGAATGTCAA

CAGAGCAAGGAGAAAAAAGGCAGTCCTGGAATCACATTCTTAGCACACCTACACCTCT

TGAAAATAGAACAACTTGCAGAATTGAGAGTGATTCCTTTCCTAAAAGTGTAAGAAAG

CATAGAGATTTGTTCGTATTTAGAATGGGATCACGAGGAAAAGAGAAGGAAAGTGATT

TTTTTCCACAAGATCTGTAATGTTATTTCCACTTATAAAGGAAATAAAAAATGAAAAA

CATTATTTGGATATCAAAAGCAAATAAAAACCCAATTCAGTCTCTTCTAAGCAAAATT

GCTAAAGAGAGATGAACCACATTATAAAGTAATCTTTGGCTGTAAGGCATTTTCATCT

TTCCTTCGGGTTGGCAAAATATTTTAAAGGTAAAACATGCTGGTGAACCAGGGGTGTT

GATGGTGATAAGGGAGGAATATAGAATGAAAGACTGAATCTTCCTTTGTTGCACAAAT

AGAGTTTGGAAAAAGCCTGTGAAAGGTGTCTTCTTTGACTTAATGTCTTTAAAAGT
A
T

_ _ CCAGAGATACTACAATATTAACATAAGAAAAGATTATATATTATTTCTGAATCGAGAT

GTCCATAGTCAAATTTGTAAATCTTATTCTTTTGTAATATTTATTTATATTTATTTAT

GACAGTGAACATTCTGATTTTACATGTAAAACAAGAAAAGTTGAAGAAGATATGTGAA

GAAAAATGTATTTTTCCTAAATAGAAATAAATGATCCCATTTTTTGGT

ORF Start: ATG at 13 ORF Stop: TAG
at 1069 SEQ ID NO: 26 (3S2 as ~MW at 38279.8kD

NOVBb, MSSLSGDCGLPPDVPNAQPALEDVQVPEDTVITYKCEESFVKIPGEKDSVICLKGSQW

PIOtelri SequeriCeLQ~'KWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSF

CLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIG

EHSIYCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPTTVNVPTTEVSPTSQKTTTK

TTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTM

GLLT

SEQ ID NO: 27 978 by NOVBC, GGATCCGACTGTGGCCTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGCC

DNA

SequeriCe ~TTCCTGGCGAGAAGGACTCAGTGATCTGCCTTAAGGGCAGTCAATGGTCAGATATT

GAAGAGTTCTGCAATCTCGGTACTGTTGTGGAATATGAGTGCCGTCCAGGTTACAGAA

GAGAACCTTCTCTATCACCAAAACTAACTTGCCTTCAGAATTTAAAATGGTCCACAGC

AGTCGAATTTTGTAAAAAGAAATCATGCCCTAATCCGGGAGAAATACGAAATGGTCAG

ACTGATGTACCAGGTGGCATATTATTTGGTGCAACCATCTCCTTCTCATGTAACACAG

GGTACAAATTATTTGGCTCGACTTCTAGTTTTTGTCTTATTTCAGGCAGCTCTGTCCA

GTGGAGTGACCCGTTGCCAGAGTGCAGAGAAATTTATTGTCCAGCACCACCACAAATT

GACAATGGAATAATTCAAGGGGAACGTGACCATTATGGATATAGACAGTCTGTAACGT

ATGCATGTAATAAAGGATTCACCATGATTGGAGAGCACTCUATTTATTGTAW
UzTGAH

TAE1T('~~lTGi-~AGGAGAGTGC~AG~t GGCCCAt~CACC T csAATGI:AGAGGAAAATCTCTAACT

TCCAAGGTCCCACCAACAGTTCAGAAACCTACCACAGTAAATGTTCCAACTACAGAAG

TCTCACCAACTTCTCAGAAAACCACCACAAAAACCACCACACCAAATGCTCAAGCAAC

ACGGAGTACACCTGTTTCCAGGACAACCAAGCATTTTCATGAAACAACCCCAAATAAA

GGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTATCTGGGCACACGTGTTTCACGT

TGACAGGTTTGCTTGGGACGCTAGTAACCATGGGCTTGCTGACTCTCGAG

ORF Start: at 7 ORF Stop:
at 973 SEQ ID NO: 28 322 as MW at 34931.OkD

NOVBC, DCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEE

PTOteiri SequenceVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDN

GIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSLTSK

VPPTVQKPTTVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGS

GTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

SEQ ID NO: 29 978 by NOV$(1, . GGATCCGACTGTGGCCTTCCCCCAGATGTACCTAATGCCCAGCCAGCTTTGGAAGGCC

Sequence AATTCCTGGCGAGAAGGACTCAGTGATCTGCCTTAAGGGCAGTCAATGGTCAGATATT

Sequence comparison of the above .protein sequences yields the following sequence relationships shown in Table 8B.
Table 8B. Comparison of NOVBa against NOVBb through NOVBd.
Protein SequenceNOYBa Residues/Identities!

Match ResiduesSimilarities for the Matched Region NOVBb 34..284 215/317 (67%) ~ 6..318 219/317 (68%) NOVBc X35..284 ~192/316(600~) 288 ~ i77i316 1 6i%

. . ( .. ) NOVBd 35..302 196/334 (58%) 3..308 201/334 (59%) Further analysis of the NOVBa protein yielded the following properties shown in Table 8C.
Table 8C. Protein Sequence Properties NOVBa PSort 0.7571 probability located in outside; 0.1000 probability located in endoplasmic analysis: reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen);
0.1000 probability located in lysosome (lumen) SignalP Cleavage site between residues 35 and 36 analysis:

A search of the NOV8a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 8D.
Table 8D. Geneseq Results for NOVBa NOVBa Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAW73505 Decay accelerating factor 1..318 300/381 (78%)e-165 protein -Homo Sapiens, 38I aa. [JP10313865-A,1..381 301/381 (78%) 02-DEC-1998]

AAY31740 Human CD55 and 791Tgp72 1..318 300/381 (78%)[ e-165 tumour z Z/~ , ma, associated antigen - Homo I ..~ moi~
sapiens, 381 81 J~ 11 J V
1 ', V .
V J

aa. [W09943800-A1, 02-SEP-1999]

AAR66683 Decay accelerating factor- 1..318 300/381 (78%)e-165 Homo sapiens, 381 aa. [IJS5374548-A,1..381 301/381 (78%) DEC-1994]

AAW27483 Human glycophosphatidylinositol1..307 282/370 (76%)e-154 anchored DAF - Homo sapiens,1..370 284/370 (76%) 440 aa.

[W09735886-Al, 02-OCT-1997]

AAR66684 Decay accelerating factor 1..307 282/370 (76%)e-154 - Homo Sapiens, 440 aa. [US5374548-A,1..370 284/370 (76%) DEC-1994]

In a BLAST search of public sequence datbases, the NOV 8a protein was found to have homology to the proteins shown in the BLASTP data in Table, 8E.
Table 8E. Public BLASTP Results for NOVBa NOV8a Identities/

Protein Residues/ SimilaritiesExpect for Accession Protein/Organism/Length Match the Matched Value Number Residues Portion CAC07712 SEQUENCE 1 FROM PATENT 1..318 300/381 (78%) e-165 W09943800 - Homo Sapiens 1..381 301/381 (78%) (Human), 3 81 aa.

P08174 Complement decay-accelerating1..318 299/381 (78%) e-164 factor precursor (CDSS antigen) 1..381 300/381 (78%) - Homo sapiens (Human), 381 aa.

CAA03840 SEQUENCE I FROM PATENT 1..307 282/370 (76%) e-153 WO9735886 - unidentified, 1..370 284/370 (76%) 440 aa.

Q9MYJ5 DECAY-ACCELERATING FACTOR 35..318 241/307 (78%) e-136 - Pan troglodytes (Chimpanzee), 305 as 1..305 251/307 (81%) (fragment).
CAC39504 SEQUENCE 31 FROM PATENT 35..294 242/323 (74%) e-130 W00132901 - unidentified, 611 aa. 289..611 243/323 (74%) PFam analysis predicts that the NOVBa protein contains the domains shown in the Table 8F.
Table 8F. Domain Analysis of NOVBa Identities/

Pfam DomainNOVBa Match RegionSimilarities. Expect Value for the Matched Region sushi 36..94 14/67 (21%) 1.4e-1 45/67 (67%) sushi 98..158 18/67 (27%) 1.6e-09 40/67 (60%) sushi 163..220 24/64 (38%) ' 8.8e-13 45/64 (70%) Example 9.
The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.
Table 9A. NOV9 Sequence Analysis ~ SEQ ID NO: 31 ~ 1266 Up _ _____._~
CG9463O-OI DNA 'GGGCGGGCTCCCACTCCATGAGGTATTTCAGCACCGCCGTTTCCTGGCCGGGCCGCGG
Sequence 'jGGAGCCCAGCTTCATTGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGGTCGAC
r,,..,..T
~.~,r.r~r.mr.TP.mnmnnr_r_~rmr_nar_rrr_~r_r_r_nr_rr~mr_r~mr~r~arc~ar..ra~c~hc';( '~
CGGAGTATTGGGACCTACAGACACTGGGCGCCAAGGCCCAGGCACAGACTGACCGAGT
GAACCTGCGGACCCTGCTCCGCTACTACAACCAGAGCGAGGCGGGGTATCACATCCTC
CAGGGAATGTTTGGCTGCGACCTGGGGCCCGACGGGCGTCTCCTCCGCGGGTATGAGC
AGTATGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCCTGGAC
GAGCAAAGGAGAGCCTACCTGGAGGGCACCTGCATGGAGTGGCTCCGCAGACACCTGG
CTACCCTGCGGAGATCACATTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGAC
CTGTGATGTGGAGGAAGAAGAGCTCAGATAGAAAAGGAGGGAGCTACTCTCAGGCTGC

WO 02/090504 PCT/US02/14342 .
AATTTCCCTCCCTTGACTCCATCAACATCGGCACCTGCCAGACGCCCACCACCCACCA

TCGAAGTGCTGAGAAGAAGTGCAAGGTACTCAACCTGCTCTGGGGATACAGCAGGAAA

GCAGAGTGTTTACGGATTTCACATTCCATCAAAGAAAATCCATTTTGA

ORF Start: ATG at 1 ORF Stop:
TGA at 1264 SEQ ID NO: 32 421 as MW at 47476.7kD

NOV9a, MAPRTLLLLLSGTLALAETWAGSHSNB2YFSTAVSWPGRGEPSFIAVGYVDDTQFVR.VD

Protein SequenceQGMFGCDLGPDGRLLRGYEQYAYDGKDYIALNEDLRSWTAADTAAQITQRKYEAANVA

EQRRAYLEGTCMEWLRRHLENGKETLQRAGITRSWVLGFYPAEITLTWQRDGEDQTQD

MELVETRPTGDGTFQKWAVVVVPSGEEQRYTCHVQHKGLPKPLILRWEPSPQPTIPIV

GIIAGLVLLGAVVTGAVVTAVMWRKKSSDRKGGSYSQAAKNIIKVKTEKFLALWCIRS

RCKLVQPAALGLRVA12DSFEFPS?~DSINIGTCQTPTTHHRSAEKKCKVLNLLWGYSRK

AECLRISHSIKENPF

Further analysis of the NOV9a protein yielded the following properties shown in Table 9B.
Table 9B. Protein Sequence Properties NOV9a PSort 0.4600 probability located in plasma membrane; 0.1335 probability located in analysis: microbody (peroxisome); O.I000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen) SignalP ~ Cleavage site between residues I8 and 19 analysis:
A search of the NOV9a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 9C.
Table 9C. Geneseq Results for NOV9a NOV9a Identities/

Geneseq Protein/Organisr.~./Length ~ Residues/Simi:ar idea ~ Eapect ~Da:ent #, for IdentifierBatej Match the Matched Value ResiduesRegion AAB36874 MHC class I protein - Unidentified,1..331 2661347 (76%)e-153 aa. [US6140305 A, 31-OCT-2000]~ 285/347 (81%) 4..350 AAB58683 HLA-A2/A28 protein #4 - 1..331 265/347 (76%)e-153 Unidentified, 365 aa. [L1S6153408-A, 28-NOV-2000]4..350 285/347 (81%) AAY52922 HLA-A2/A28 family peptide 1..331 265/347 (76%)e-153 A2 (Lee) SEQ ID NO:100 - Mammalia, 4..350 285/347 (81%) 365 aa.

[US5976551-A, 02-NOV-1999]

AAY68268 Human leukocyte antigen I ..331 265/347 (76%)e-153 A2/A28 family protein SEQ ID NO:100 - 4..350 285/347 (81 Homo sapiens, %) 365 aa. [US60I 1146-A, 04-JAN-2000]

AAB58687 HLA-A2/A28 protein #8 - 1..331 264/347 (76%)e-153 Unidentified, 365 aa. [US6153408-A, 28-NOV-2000]4..350 284/347 (81%) In a BLAST search of public sequence datbases, the NOV9a protein was found to have homology to the proteins shown in the BLASTP data in Table 9D.
Table 9D. Public BLASTP Results for NOV9a NOV9a Identities/

Protein Residues/SimilaritiesExpect for AccessionProtein/Organism/Length Match the Matched Value Number ResiduesPortion I54493 MHC class I histocompatibility1..331 273/347 (78%),e-158 antigen HLA-A alpha chain precursor4..350 292/347 (83%) - human, 365 aa.

Q9MXI8 MHC CLASS I ANTIGEN - Pan 1..331 274/347 (78%)e-158 troglodytes (Chimpanzee), 4..350 292/347 (83%) 365 aa.

Q9MXL0 MHC CLASS I ANTIGEN - Pan 1..331 274/347 (78%)e-I 58 troglodytes (Chimpanzee), 4..350 291/347 (82%) 365 aa.

Q9MXI7 MHC CLASS I ANTIGEN - Pan 1..331 274/347 (78%)e-158 troglodytes (Chimpanzee), 2..348 291/347 (82%) 363 as (fragment).

Q9TPL3 MHC CLASS I ANTIGEN I ..331 274/347 (78%)e-158 (LYMPHOCYTE ANTIGEN) - Pan 4..350 290/347 (82%) troglodytes (Chimpanzee), 365 aa.

PFam analysis predicts that the NOV9a protein contains the domains shown in the Table 9E.
Table 9E.
Domain Analysis of NOV9a Identities/

Pfam DomainNOV9a Match RegionSimilarities Expect ' Value for the Matched Region MHC_I 22..200 140/180 (78%) 3.4e-131 170/180 (94%) ig 212..266 12/56 (21%) 0.00049 41/56 (73%) Example 10.
The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 10A.

----..~.....
Table 10A.
NOV10 Sequence Analysis SEQ ID NO: 33 717 by NOVlOa, GCAGCATGGGGAGCTTCCACGCGGGCATACGGTGCATCAAGTACATGCTGGTTGGCTT

CG94831-O1 C~CCTGCTCTTCTGGCTGGCTGGATCGGCCGTCATTGCTTTTGGACTATGGTTTCGG
DNA

Sequence TTCGGAGGTGCCATAAAGGAGTTATCATCAGAGGACAAGTCCCCAGAGTATTTCTATG

TGGGTGGGCTGTATGTTCTGGTTGGAGCCGGGGCCCTGATGATGGCCGTGGGGTTCTT

CGGGTGCTGCGGAGCCATGCGGGAGTCGCAATGTGTGCTTGGATCATTTTTTACCTGC

CTCCTGGTGATATTTGCTGCTGAAGTAACCACTGGAGTATTTGCTTTTATAGGCAAGG

CTATCCGACATGTTCAGACCATGTATGAAGAGGCTTACAATGATTACCTTAAAGACAG

GGGAAAAGGCAATGGGACACTCATCACCTTCCACTCAACATTTCAGTGCTGTGGAAAA

GAAAGCTCCGAACAGGTCCAACCTACATGCCCAAAGGAGCTTCTAGGACACAAGAATT

GCATCGATGAAATTGAGACCATAATCAGTGTTAAGCTCCAGCTCATTGGAATTGTCGG

TATTGGAATTGCAGGTCTGACGGTGATCTTTGGCATGATATTCAGCATGGTCCTCTGC

TGTGCGATACGAAACTCACGAGATGTGATATGAAGCTACTTCTACATGAAAATTGCAA

TCTAAAGCTTTCATACCAAAT

ORF Start: ATG at 6 ORF Stop: TGA
at 669 SEQ ID NO: 34 X221 as MW at 24077.1kD

NOVlOa, MGSFHAGIRCIKYMLVGFNLLFWLAGSAVIAFGLWFRFGGAIKELSSEDKSPEYFYVG

Protein Sequence~Q~EAYNDYLKDRGKGNGTLITFHSTFQCCGKESSEQVQPTCPKELLGHKNCI

DEIETIISVKLQLIGIVGIGIAGLTVIFGMIFSMVLCCAIRNSRDVI

SEQ ID NO: 35 742 by NOVIOb, TGTAGGTCTTCTTGATCCGCAGCATGGGGCGCTTCCGCGGGGGCCTGCGGTGCATCAA

DNA

Sequence TTTGGACTATGGTTTCGGTTCGGAGGTGCCATAAAGGAGTTATCATCAGAGGACAAGT

CCCCAGAGTATTTCTATGTGGGGCTGTATGTTCTGGTTGGAGCCGGGGCCCTGATGAT

GGCCGTGGGGTTCTTCGGATGCTGCGGAGCCATGCGGGAGTCGCAATGTGTGCTTGGA

TCATTTTTTACCTGCCTCCTGGTGATATTTGCTGCTGAAGTAACCACTGGAGTATTTG

CTTTTATAGGCAAGGGGGTAGCTATCCGACATGTTCAGACCATGTATGAAGAGGCTTA

CAATGATTACCTTAAAGACAGGGGAAAAGGCAATGGGACACTCATCACCTTCCACTCA

ACATTTCAGTGCTGTGGAAAAGAAAGCTCCGAACAGGTCCAACCTACATGCCCAAAGG

AGCTTCTAGGACACAAGAATTGCATCGATGAAATTGAGACCATAATCAGTGTTAAGCT

CCAGCTCATTGGAATTGTCGGTATTGGAATTGCAGGTCTGACGATCTTTGGCATGATA

TTCAGCATGGTCCTCTGCTGTGCGATACGAAACTCACGAGATGTGATATGAAGCTACT

TCTACATGAAAATTGCAATCTAAAGCTTTCATACACAAATAAGGGC

OItF Start: ATG at 24 ORi StnY: T O
A at 687 SEQ ID NO: 36 221 MW at 24147.2kD
as NOVlOb, MGRFRGGLRCIKYLLLGFNLLFWLAGSAVIAFGLWFRFGGAIKELSSEDKSPEYFYVG

Protein SequenceT~Q~'M~~~~~RGKGNGTLITFHSTFQCCGKESSEQVQPTCPKELLGHKNC

IDEIETIISVKLQLIGIVGIGIAGLTIFGMIFSMVLCCAIRNSRDVI

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 10B.
Table 10B. Comparison of NOVlOa against NOVlOb.
Protein Sequence NOVlOa Residues/ Identities/
Match Residues Similarities for the Matched Region NOV 1 Ob 1..221 ~ 194/223 (86%) 1..221 195/223 (86%) Further analysis of the NOV 10a protein yielded the following properties shown in Table 10C.
Table 10C. Protein Sequence Properties NOVlOa PSort 0.6400 probability located in plasma membrane; 0.4000 probability Located in GoIgi analysis: body; 0.3000 probability located in endoplasmic reticulum (membrane); 0.0300 probability located in mitochondria) inner membrane SignalP Cleavage site between residues 42 and 43 analysis:
A search of the NOV 10a 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 OD.
Table )OD. Geneseq Results for NOVlOa NOVlOa Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the MatchedValue ResiduesRegion AAG73745 Human colon cancer antigen 1..221 213/223 e-119 protein SEQ (95%) ID N0:4509 - Homo sapiens, 9..229 216/223 229 aa. (96%) [W0200122920-A2, OS-APR-2001]

AAW61623 Clone HAIDQ59 5'of TM4SF 1..221 213/223 e-119 (95%) superfamily - Homo sapiens,1..221 216/223 221 aa. (96%) [W09831799-A2, 23-JUL-1998]

ABB57234 Mouse ischaemic condition 7..221 103/223 2e-50 related (46%) protein sequence SEQ ID 6..226 137/223 N0:6I2 - Mus (6I%) . . ., musculus, 226 aa. [W0200188188-A2, ABB44580 Mouse wound healing related7..221 103/223 2e-50 polypeptide (46%) SEQ 1D NO 37 - Mus musculus,6..226 1371223 226 aa. (61%) [CA2325226-A 1, 17-MAY-2001 ]

AAG75156 Human colon cancer antigen 7..221 103/225 1 e-49 protein SEQ (45%) ID N0:5920 - Homo Sapiens, 53..275 137/225 275 aa. (60%) [W0200122920-A2, OS-APR-2001]

In a BLAST search of public sequence datbases, the NOV 1 Oa protein was found to have homology to the proteins shown in the BLASTP data in Table 10E.

Table 10E. Public BLASTP Results for NOVlOa NOVlOa Protein Identities/

Accession Protein/Organism/Length Residues/Similarities Expect for the Number Matched PortionValue Residues Q8WU05 HYPOTHETICAL 24.1 KDA I..221 213/223 (95%) e-I

PROTEIN - Homo sapiens 1..221 216/223 (96%) (Human), 221 aa.

Q9JJW 1 TSPAN-2 PROTEIN - Rattus 1..221 202/223 (90%) e-114 norvegicus (Rat), 221 1..221 213/223 (94%) aa.

Q922J6 RIKEN CDNA 6330415F13 1..221 200/223 (89%) e-113 GENE -Mus musculus (Mouse), 1..221 210/223 (93%) 221 aa.

Q9D39'7 6330415F13RIK PROTEIN 1..221 199/223 (89%) e-113 - Mus musculus (Mouse), 221 1..221 ~
aa. 210/223 (93%) 060636 Tetraspanin 2 (Tspan-2) 1..221 206/224 (91%) e-112 - Homo ' Sapiens (Human), 222 aa. 1..222 209/224 (92%) PFam analysis predicts that the NOV 1 Oa protein contains the domains shown in the Table 1 OF.
Table 10F. Domain Analysis of NOVlOa Identities/
Pfam Domain NOVlOa Match Region Similarities Expect Value for the Matched Region transmembrane4 12..214 68/268 (25%) S.l e-62 168/268 (63%) Examule 11.
The NOV 11 clone was analyzed, and the nucleotide and encoded polypeptide sequences are showmin Table 1 1A.

CACCTGTCATAAAATCCAGTGGAAGATTTCTATGGATTAAATTTTTTGCTGATGGAGA
GACCTTGGAGCTTTGAAACCATTACCAGCGTGTGAGTTTGAGATGGGCGGTTCCGAAG
CATGCTACGCACGGGTCTTGGGGTGATCCGCATGTGGGCAGATGAGGGCAGTCGAAAC
AGCCGATTTCAGATGCTCTTCACATCCTTTCAAGAACCTCCTTGTGAAGGCAACACAT
TCTTCTGCCATAGTAACATGTGTATTAATAATACTTTGGTCTGCAATGGACTCCAGAA
GAGTTATGCACTCTCAGAGGGACAGGAGCTACAGCTGACTTTGCAGATGTGGCAGATG
ACTTTGAAAATTACCATAAACTGCGGAGGTCATCTTCCAAATGCATTCATGACCATCA
CTGTGGATCACAGCTGTCCAGCACTAAAGGCAGCCGCAGTAACCTCAGCACAAGAGAT
GCTTCTATCTTGACAGAGATGCCCACACAGCCAGGAAAACCCCTCATCCCACCCATGA
ACAGAAGAAATATCCTTGTCATGAAACACAGCTACTCGCAAGATGCTGCAGATGCCTG
TGACATAGATGAAATCGAAGAGGTGCCGACCACCAGTCACAGGCTGTCCAGACACGAT
OIZF Start: ATG at 98 ORF Stop:~TGA at 1676 SEQ ID NO: 38 526 as MW at 59333.81cD
NOVIIa, MIHGRSVLHIVASLIILHLSGATKKGTEKQTTSETQKSVQCGTWTKHAEGGIFTSPNY
CG94892-Ol PSKYPPDRECIYIIEAAPRQCIELYFDEKYSIEPSWECKFDHIEVRDGPFGFSPIIGR
Protein Sequence FCGQQNPPVIKSSGRFLWIKFFADGELESMGFSARYNFTPDPDFKDLGALKPLPACEF
EMGGSEGIVESIQIMKEGKATASEAVDCKWYIRAPPRSKIYLRFLDYEMQNSNECKRN
FVAVYDGSSSVEDLKAKFCSTVANDVMLRTGLGVIRMWADEGSRNSRFQMLFTSFQEP
PCEGNTFFCHSNMCINNTLVCNGLQNCVYPWDENHCKEKRKTSLLDQLTNTSGTVIGV

FADVADDFENYHKLRRSSSKCIHDHHCGSQLSSTKGSRSNLSTRDASILTEMPTQPGK
PLIPPMNRRNILVMKHSYSQDAADACDIDEIEEVPTTSHRLSRHDKAVQRSVSIDFLM
TTIT
Further analysis of the NOV 1.1 a protein yielded the f~iloWing pr~pert:es sho~,hrr: in ?'able 11B.
Table I1B. Protein Sequence Properties NOVlla Psort 0.4600 probability located in plasma membrane; 0.1000 probability located in analysis: endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); O.I000 probability located in outside SignaIP ~ Cleavage site between residues 23 and 24 analysis:
A search of the NOV 11 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 11 C.

Table 11C. Geneseq Results for NOVlla NOVlIa Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAB47296 PR04401 polypeptide - Homo 10..520 301 /519 (57%)e-180 Sapiens, 525 aa. [W0200140465-A2, 14..525 387/519 (73%) 2001]

AAU12228 Human PR04401 polypeptide 10..520 3011519 (S7%)e-180 sequence -Homo Sapiens, S2S aa. [W0200140466-14..525 387/519 (73%) A2, 07-JUN-2001 J

AAM93946 Human polypeptide, SEQ ID 10..520 301/519 (57%)e-180 NO: 4135 -Homo Sapiens, S2S aa. [EP1130094-A2,14..525 387/519 (73%) AAU 18670Renal and cardiovascular-associated66..520 282/463 (60%)e-16t"s protein, Seq ID 109 - Homo 19..474 356/463 (7S%) Sapiens, 474 ,~

aa. [W0200155328-A2, 02-AUG-2001]

ABBSS774 Human polypeptide SEQ ID 133..520234/396 (S9%)e-135 Homo Sapiens, 389 aa. [L152001039335-1..389 296/396 (74%) A 1, 08-NOV-2001 J

In a BLAST search of public sequence datbases, the NOV 11 a protein was found to have homology to the proteins shown in the BLASTP data in Table 11 D.
Table 11D. Public BLASTP
Results for NOVlla Protein NOVlIa Identities/

Accession Protein/Organism/Length Residues/~ Expect Similarities ' for Number . _ _ . Match ; the Matchedvalve ResiduesPortion AAL48461 GH11189P - Drosophila 55..363 107/318 (33%)1e-47 melanogaster (Fruit fly), 677 aa. 154..448163/318 (SO%) Q96SP4 CDNA FLJ14724 FIS, CLONE 327..52091/201 (45%) 9e-41 NT2RP3001716 - Homo sapiens8..201 130/201 (64%) (Human), 201 aa.

061 849 K03E5.1 PROTEIN - Caenorhabditis52..287 82/246 (33%) 3e-32 elegans, 321 aa. 75..31 ~
S 126/246 (S0%) Q96RU9 INTRINSIC FACTOR-VITAMIN 37..295 77/265(29%) 3e-28 B 12 RECEPTOR - Homo sapiens2084..2326138/265 (S2%) (Human), 3494 as (fragment).

060494 INTRINSIC FACTOR-B 12 37..295 77/265 (29%) 3e-28 .

RECEPTOR PRECURSOR - Homo2213..2455138/265 (52%) Sapiens (Human), 3623 aa.

PFam analysis predicts that the NOV 11 a protein contains the domains shown in the Table 11 E.
Table 11E. Domain Analysis of NOVlla Identities/

Pfam DomainNOVlla Match Similarities Expect Region Value for the Matched Region CUB 41..152 43/I 18 (36%) 6.3e-29~

84/118 (71%) CUB 172..284 27/124 (22%) 0.0003 64/124 (52%) ldl_recept 290..328 13/43 (30%) 0.00073 a 2 5/43 (58%) Example 12.
The NOV 12 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 12A.
Table 12A. NOV12 Sequence Analysis ( ~SEQ ID NO: 39 X2302 by NOV 12a, Sequence GCCCCTCAGGCATTACTATCCCTGGAAAACCAGGTGCCCAAGGGGTGCCAGGGCCCCC
CTCAAGGGGGATAATGGAGTGGGCCAGCCCGGGCTGCCTGGGGCCCCAGGGCAGGGGG
TGGTCCCCCAGGGCTCCCTGGCAAGGTCGGGCCACCAGGGCAGCCGGGGCTTCGGGGG
GAGCCAGGAATACGAGGGGACCAGGGCCTCCGGGGACCCCCAGGACCCCCTGGCCTCC
CGGGCCCCTCAGGCATTACTATCCCTGGAAAACCAGGTGCCCAAGGGGTGCCAGGGCC
GGCCTCAAGGGGGATAATGGAGTGGGCCAGCCCGGGCTGCCTGGGGCCCCAGGGCAGG
GGGGTGCCCCCGGCCCCCCCGGCCTCCCTGGTCCAGCTGGCTTAGGCAAACCTGGTTT
ACAGGGGTGAGCCAGGGGAGGATGGGGAGCCAGGGGAGCAGGGCCCACAGGGTCTTGG
CCTAAGGGTGAGGCAGGGCCTGGAGGACCCCCAGGAGTGCCTGGCATTCGAGGTGACC
GGCCCATGGACCCCCTGGACCAACTGGGCCCAAGGGTGAGCCGGGTTTCACGGGTCGC
CCTGGAGGACCAGGGGTGGCAGGAGCCCTGGGGCAGAAAGGTGACTTGGGGCTCCCTG
GGCAGCCTGGCCTGAGGGGTCCCTCAGGAATCCCAGGACTCCAGGGTCCAGCTGGCCC
TATTGGGCCCCAAGGCCTGCCGGGCCTGAAGGGGGAACCAGGCCTGCCAGGGCCCCCT

GGAGAGGGGAGAGCAGGGGAACCTGGCACGGCTGGGCCCACGGGGCCCCCAGGGGTCCi CTGGCTCCCCTGGAATCACGGGCCCTCCGGGGCCTCCCGGGCCCCCGGGACCCCCTGG

TGCCCCTGGGGCCTTCGATGAGACTGGCATCGCAGGCTTGCACCTGCCCAACGGCGGT

GTGGAGGGTGCCGTGCTGGGCAAGGGGGGCAAGCCACAGTTTGGGCTGGGCGAGCTGT

CTGCCCATGCCACACCGGCCTTCACTGCGGTGCTCACCTCGCCCTTCCCCGCCTCGGG

CATGCCCGTGAAATTTGACCGGACTCTCTACAATGGCCACAGCGGCTACAACCCAGCC

ACTGGCATCTTCACCTGCCCTGTGGGCGGCGTCTACTACTTTGCTTACCATGTGCACG

TCAAGGGCACCAACGTGTGGGTGGCCCTGTACAAGAACAACGTGCCGGCCACCTATAC

CTACGATGAGTACAAGAAGGGCTACCTGGACCAGGCATCTGGTGGGGCCGTGCTCCAG

CTGCGGCCCAACGACCAGGTCTGGGTGCAGATGCCGTCGGACCAGGCCAACGGCCTCT

ACTCCACGGAGTACATCCACTCCTCCTTTTCAGGATTCTTGCTCTGCCCCACATAA
CC

_ CGCGGGGGGGGTCCTGCTGCCCTGGCCTCCTCCCCTTTAGTGGTAGAGCGACCTTTTC

AATTACAAAGAACCTCCTGGp~~AAAAAAACAAAAGCTNNN

ORF Start: ATG at ORF Stop:

at 2200 SEQ ID NO: 40 733 as MW at 6999S.4kD

NOVl2a, MDYWVPPHPVIFLFLFFLVETGFHHVGQAGLKLLTSSNPPPGLPGKVGPPGQPGLRGE

Protein SequenceLKGDNGVGQPGLPGAPGQGGAPGPPGPAGPPGFSRMGKAGPPGLPGKVGPPGQPGLRG

EPGIRGDQGLRGPPGPPGLPGPSGITIPGKPGAOGVPGPPGFOGEPGPOGEPGPPGDR

GLKGDNGVGQPGLPGAPGQGGAPGPPGLPGPAGLGKPGLDGLPGAPGDKGESGPPGVP

GPRGEPGAVGPKGPPGVDGVGVPGAAGLPGPQGPSGAKGEPGTRGPPGLIGPTGYGMP

GLPGPKGDRGPAGVPGLLGDRGEPGEDGEPGEQGPQGLGGPPGLPGSAGLPGRRGPPG

PKGEAGPGGPPGVPGIRGDQGPSGLAGKPGVPGERGLPGAHGPPGPTGPKGEPGFTGR

PGGPGVAGALGQKGDLGLPGQPGLRGPSGIPGLQGPAGPIGPQGLPGLKGEPGLPGPP

GEGRAGEPGTAGPTGPPGVPGSPGITGPPGPPGPPGPPGAPGAFDETGIAGLHLPNGG

VEGAVLGKGGKPQFGLGELSAHATPAFTAVLTSPFPASGMPVKFDRTLYNGHSGYNPA

TGIFTCPVGGVYYFAYHVHVKGTNVWVALYKNNVPATYTYDEYKKGYLDQASGGAVLQ

LRPNDQVWVQMPSDQANGLYSTEYIHSSFSGFLLCPT

SEQ ID NO: 4I 1950 by NOVl2b, TGACTGCCCCTTTCTCTTTCTTTCTCAGAAATGCCTCTACCGCTGCTGCCGATGGACC

DNA

Sequence TGGCTTCCCAGGAAAACCAGGCCATGGAAAGCCAGGACTCCATGGGCAGCCTGGCCCT

GCTGGGCCCCCTGGCTTCTCCCGGATGGGCAAGGCTGGTCCCCCAGGGCTCCCTGGCA

ACGTCGGGCCACCAGGGCAGCCGGGGCTTCGGGGGGAGCCAGGAATACGAGGGGACCA

GGGCCTCCGGGGACCCCCAGGACCCCCTGGCCTCCCGGGCCCCTCAGGCATTACTATC

CCTGGAAAACCAGGTGCCCAAGGGGTGCCAGGGCCCCCAGGATTCCAGGGGGAACCAG

GGCCCCAGGGCGAGCCTGGGCCCCCAGGTGATCGAGGCCTCAAGGGGGATAATGGAGT

GGGCCAGCCCGGGCTGCCTC~GGCCC~,.AGGGCAGGGGGGTGCCCCCGGCCCCCCCGGC

CTCCCTGGTCCAGC T
GGCTTAGGCAAACC T
GGTTTGGA'i'GGG W
WCC~i~i;GGGi:CCC:AG

GAGACAAGGGTGAGTCTGGGCCTCCTGGAGTTCCAGGCCCCAGGGGGGAGCCAGGAGC

TGTGGGCCCAAAAGGACCTCCTGGAGTAGACGGTGTGGGAGTCCCAGGGGCAGCAGGG

TTGCCAGGACCACAGGGCCCATCAGGGGCCAAAGGGGAGCCAGGAACCCGGGGCCCCC

CTGGGCTGATAGGCCCCACTGGCTATGGGATGCCAGGACTGCCAGGCCCCAAGGGGGA

CAGGGGCCCAGCTGGGGTCCCAGGACTCTTGGGGGACAGGGGTGAGCCAGGGGAGGAT

GGGGAGCCAGGGGAGCGGGGCCCACAGGGTCTTGGGGGTCCCCCCGGACTTCCTGGGT

CTGCAGGGCTTCCTGGCAGACGTGGGCCCCCTGGGCCTAAGGGTGAGGCAGGGCCTGG

AGGACCCCCAGGAGTGCCTGGCATTCGAGGTGACCAGGGGCCTAGTGGCCTGGCTGGG

AAACCAGGGGTCCCAGGTGAGAGGGGACTTCCTGGGGCCCATGGACCCCCTGGACCAA

CTGGGCCCAAGGGTGAGCCGGGTTTCACGGGTCGCCCTGGAGGACCAGGGGTGGCAGG

AGCCCTGGGGCAGAAAGGTGACTTGGGGCTCCCTGGGCAGCCTGGCCTGAGGGGTCCC

TCAGGAATCCCAGGACTCCAGGGTCCAGCTGGCCCTATTGGGCCCCAAGGCCTGCCGG

G,CCTGAAGGGGGAACCAGGCCTGCCAGGGCCCCCTGGAGAGGGGAGAGCAGGGGAACC

TGGCACGGCTGGGCCCACGGGGCCCCCAGGGGTCCCTGGCTCCCCTGGAATCACGGGC

CCTCCGGGGCCTCCCGGGCCCCCGGGACCCCCTGGTGCCCCTGGGGCCTTCGATGAGA

CTGGCATCGCAGGCTTGCACCTGCCCAACGGCGGTGTGGAGGGTGCCGTGCTGGGCAA

GGGGGGCAAGCCACAGTTTGGGCTGGGCGAGCTGTCTGCCCATGCCACACCGGCCTTC

ACTGCGGTGCTCACCTCGCCCTTCCCCGCCTCGGGCATGCCCGTGAAATTTGACCGGA

CTCTCTACAATGGCCACAGCGGCTACAACCCAGCCACTGGCATCTTCACCTGCCCTGT

relationships shown in Table 12B.
Table 12B. Comparison of NOVl2a against NOVl2b.
Protein Sequence ~ NOVl2a Residues/ Identities/
Match Residues Similarities for the Matched Region NOVl2b ~ 565..733 ~ 155/169 (91%) 470..638 156/169 (91%) Further analysis of the NOV 12a~ protein yielded the following properties shown in Table 12C.
Table 12C. Protein Sequence Properties NOVl2a PSort 0.5899 probability located in outside; 0.1000 probability located in endoplasmic analysis: reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen);
0.1000 probability located in lysosome (lumen) SignaIP - Cleavage site between residues 22 and 23 analysis:
A search of the NOV 12a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 12D.

12?
Sequence comparison of the above protein sequences yields the foiiowing sequence Table 12D. Geneseq Results for NOVl2a NOVl2a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesEzpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAM79782 Human protein SEQ ID NO 96..733 607/640 (94%)0.0 Homo sapiens, 644 aa. 16..644 609/640 (94%) [W0200157190-A2, 09-AUG-2001]

AAM78798 Human protein SEQ 1D NO 96..733 607/640 (94%)0.0 Homo sapiens, 635 aa. 7..635 609/640 (94%) [WO200157190-A2, 09-AUG-2001 AAM39127 Human polypeptide SEQ ID 100..732369!649 (56%)0.0 Homo sapiens, 744 aa. 117..743418/649 (63%) [W0200153312-Al, 26-JUL-2001]

AAM40913 Human polypeptide SEQ ID i 00.. 368/649 (~6io)' 0.0 Homo Sapiens, 755 aa. 128..754417/649 (63%) [W0200153312-A1, 26-JUL-2001]

AAW57673 Collagen-like polymer - ~ 24..621~ 317/629 ~ e-159 Synthetic, 829 (50%) aa. (US5773249-A, 30-JUN-1998]42..662 341/629 (53%) In a BLAST search of public sequence datbases, the NOV 12a protein was found to have homology to the proteins shown in the BLASTP data in Table 12E.
Table 12E. Public BLASTP Results for NOVl2a Protein ~ NOVl2aIdentities/

AccessionProtein/Organisnn/Length Residues/SimilaritiesE$pect for Match the Matched Value Number ResiduesPortion BAB84955 FLJ00201 PROTEIN - Homo 68..733 623/670 (92%)0.0 Sapiens (Human), 705 as (fragment).51..705 631/670 (93%) P25067 Collagen alpha 2(VIII) chain96..733 603/640 (94%)0.0 (Endothelial collagen) - 7..635 6081640 (94%) Homo Sapiens (Human), 635 as (fragment).

A24450 collagen alpha 2(VIII) chain96..570 422/477 (88%)0.0 - bovine, 469 as (fragment). 1..469 432/47? (90%) Q9D2V4 ~ PROCOLLAGEN, TYPE VIII, 5..732 400/760 (52%)0.0 ALPHA 1 - Mus musculus (Mouse),3..743 460/760 (59%) 744 aa.

Q92I S8 PROCOLLAGEN, TYPE VIII, 5..732 399/760 (52%)0.0 ALPHA 1 - Mus musculus (Mouse),3..743 460/760 (60%) 744 aa.

PFam analysis predicts that the NOV I2a protein contains the domains shown in the Table 12F.
Table 12T. Domain Analysis of NOVl2a Identities/

Pfam DomainNOVl2a Match Similarities Expect Region Value for the Matched Region Collagen 25..83 28/60 (47%) 0.00092 37/60 (62%) Collagen 86..144 34/60 (57%) 2.4e-05 51/60 (85%) Collagen 151..208 33/60 (55%) 0.0002 46/60 (77%) Collagen 209..267 35/60 (58%) 0.00054 47/60 (78%) Collagen 270..328 31/60 (52%) 5.2e-05 46/60 (77%) Collagen 329..387 29/60 (48%) 0.00048 44/60 (73%) Collagen 388..447 35/60 (58%) 4.4e-1 48/60 (80%) Collagen 448..507 34/60 (57%) 8.9e-1 46/60 (77%) Collagen 508..566 39/60 (65%) 9.3e-05 50/60 (83%) Clq 606..730 68/137 (50%) ~ 3.4e-75 123/137 (90%) Example 13.
S The NOV 13 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 13A.
Table 13A. NOV13 Sequence Analysis SEQ ID NO: 43 [ 789 by NOVl3a, ATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATGTGCCTGTGG
CG96384-O1 DNA ~CCTTGCCCTGGGCATCGCTGCTGCTGCCAGCGCCCTTCACCCATGCTTCAGCATGG
SeqllenCe TGCCCATACGCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAGCACAGGAGA
GATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCTTAGTGTGCT

CAGGAGATCGCTCTTCAGCAAATAATGTCTCAGACTGTGAATGTGAAAA,ACGATATGA

TTACTTTGGAGAAGAGTGAATTTTCAGCCCTCAGAGCAGAACGTGAGAAAATAAAACT

CAAACTACATCAGTTAAAACAAGTAATGGATGAAGTGATTAAAGTCCGAACAGATACT

AAATTAGACTTCAACCTAGAAAAGAGCAGAGTAAAAGAATTGTATTCGTTGAATGAAA

GGAAGCTGCTGGAATTGAGAACAGAAATAGTGACATTGCATGCCCAGCAAGATTGGGC

CGTCACCCAGAGAGATAGGAAGATAGAAACTGAGGATGCTGGCCCCAAAACCATGCTT

GAGTCATACAAGCTTGATAATATTAAATATTTAGCAGGGTCTATATTTACGTGCCTAA

CAGTAGCTCTGGGATTTTATCACCTGTGGATCTAA

ORF Start: ATG at 1 OItF Stop: TAA
at 787 SEQ ID NO: 44 262 as MW at 30251.7kD

NOVl3a, MPWVCRPTGWTKRRVDVPVGPCPGHRCCCQRPSPMLQHGAHTHFLQESAGYLQLEHRR

CG96384-O1 DFSSSGSRKLSFDTRSLVCFLEDHGFATQQAEIIVSALVQVLEANVDIVYIIC)MATKNIK

Protein SequenceQEIALQQIMSQTVNVKNDMITLEKSEFSALRAEREKIKLKLHQLKQVMDEViKVRTDT

KLDFNLEKSRVKELYSLNERKLLELRTEIVTLHAQQDWAVTQRDRKIETEDAGPKTML

ESYKLDNIKYLAGSIFTCLTVALGFYHLWI

SEQ ID NO: 45 789 by NOVl3b, ATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATGTGCCTGTGG
~

DNA

Sequence TGCCCATACGCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAGCACAGGAGA

GATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCTTAGTGTGCT

TTCTGGAAGACCATGGGTTTGCTACTCAGCAAGCAGAAATCATTGTGTCTGCATTGGT

CCAGGTACTGGAGGCCAACGTGGACATCGTCTACAAAGATATGGCCACCAAGATGAAG

CAGGAGATCGCTCTTCAGCAAATAATGTCTCAGACTGTGAATGTGAAAAACGATATGA

TTACTTTGGAGAAGAGTGAATTTTCAGCCCTCAGAGCAGAACGTGAGAAAATAAAACT

CAAACTACATCAGTTAAAACAAGTAATGGATGAAGTGATTAAAGTCCGAACAGATACT

AAATTAGACTTCAACCTAGAAAAGAGCAGAGTAAAAGAATTGTATTCGTTGAATGAAA

GGAAGCTGCTGGAATTGAGAACAGAAATAGTGACATTGCATGCCCAGCAAGATTGGGC

CGTCACCCAGAGAGATAGGAAGATAGAAACTGAGGATGCTGGCCCCAAAACCATGCTT

GAGTCATACAAGCTTGATAATATTAAATATTTAGCAGGGTCTATATTTACGTGCCTAA

CAGTAGCTCTGGGATTTTATCACCTGTGGATCTAA

ORF Start: ATG at ORF Stop: TAA at 787 SEQ ID NO: 46 262 as MW at 30251.7kD

NOVl3b, MPWVCRPTGWTKRRVDVPVGPCPGHRCCCQRPSPMLQHGAHTHFLQESAGYLQLEHRR

PrOteln SequenceQEIALQQIMSQTVNVKNDMITLEKSEFSALRAEREKIKLKLHQLKQVMDEVIKVRTDT

KLDFNLEKSRVKELYSLNERKLLELRTEIVTLHAQQDWAVTQRDRKIETEDAGPKTML

ECvKLDNIKI'LAGS T_FTCLTVALGFYUT..~WI

SEQ ID NO: 47 285 by NOV13C, GGATCCACCATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATG

DNA

Sequence TCAGCATGGTGCCTATACTCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAG

CACAGGAGAGATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCT

TAGTGTGCTTTCTGGAAGACCATGGGTTTGCTACTCAGCAAGCAGAACTCGAG

ORF Start: at 1 ORF
Stop: end of sequence SEQ ID'NO: 48 95 as MW at 10768.1kD

NOV13C, GSTMPWVCRPTGWTKRRVDVPVGPCPGHRCCCQRPSPMLQHGAYTHFLQESAGYLQLE

209749131 ~FSSSGSRKLSFDTRSLVCFLEDHGFATQQAELE
Protein Sequence .

SEQ ID NO: 49 801 by NOVl3d, GGATCCACCATGCCTTGGGTATGCCGCCCCACTGGCTGGACAAAGCGGCGCGTGGATG

DNA

Sequence TCAGCATGGTGCCTATACTCACTTCCTGCAGGAGTCTGCTGGATACCTGCAGCTGGAG

CACAGGAGAGATTTCAGCTCTTCTGGGAGTAGGAAGCTCTCCTTTGACACTCGTTCCT

relationships shown in Table 13B.
Table 13B.
Comparison of NOVl3a against NOVl3b through NOVl3d.

Protein Sequence( NOVl3a Residues/Identities/

Match ResiduesSimilarities for the Matched Region NOVl3b 1..262 262/262 (100%) 1..262 262/262 (100%) NOV 13c 1..91 89/91 (97%) 4..94 91/91 (99%) NOV 13d 1..262 261 /262 (99%) 4..265 262/262 (99%) Further analysis of the NOV 13a protein yielded the following properties shown in Table 13C.
Table 13C. Protein Sequence Properties NOVl3a PSort 0.7000 probability located in plasma membrane; 0.2000 probability located in analysis: endoplasmic reticulum (membrane); 0.1000 probability located in mitochondria) inner membrane; 0.0000 probability located in endoplasmic reticulum (lumen) SignalP ~ No Known Signal Sequence Predicted analysis:
A search of the NOV 13 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 13D.

Sequence comparison of the above protein sequences yields the following sequence Table 13D. Geneseq Results for NOVl3a NOVl3a Identities/

Geneseq Protein/Organism/Length [ Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAM40693 Human polypeptide SEQ ID [ 45..262185/220 (84%)2e-9S

Homo Sapiens, 246 aa. [WO200I27..246 197/220 (89%) AI, 26-JUL-2001) ~

AAM38907 Human polypeptide SEQ ID 104..262138/160 (86%)2e-70 Homo Sapiens, 160 aa. [W0200I53312-~ 1..160145/160 (90%) A1, 26-JUL-2001) AAG73708 Human colon cancer antigen 142..262110/122 (90%)8e-54 protein SEQ ID NO:4472 - Homo Sapiens,8..129 I 141122 129 (93%) - aa. [W0200122920-A2, OS-APR-2001]

AAG787S0 Human calthrin light chain 111..26170/1 S2 (46io)' Se-34 17 - Homo ' sapiens, 153 aa. [W020017S04S-A2,I..1S2 104/152 (68%) I I-OCT-2001 ) AAB28214 Novel human protein # 12 s 59..19767/140-(47%)2e-30 - Homo sapiens, 1 S6 aa. [WO2000S216S-A2,17..1 99/140 (69%) SEP-2000]
....

In a BLAST search of public sequence datbases, the NOV 13a protein was found to have homology to the proteins shown in the BLASTP data in Table 13E.
Table 13E. Public BLASTP
Results for NOVl3a Protein NOVl3a ~ Identities/

AccessionProtein/Organism/Length Residues/SimilaritiesExpect for Number Maxch ~ the Matched! Value Residues Portion Q96JS7 SIMILAR TO RIKEN CDNA 45..262 186/220 (84%)1e-96 6230416A05 GENE - Homo 102..321 198/220 (89%) Sapiens (Human), 321 as (fragment).

Q96AQ8 SIMILAR TO RIKEN CDNA 45..262 185/220 (84%)Se-9S

6230416A05 GENE - Homo 140..359 197/220 (89%) Sapiens (Human), 359 aa.

Q9NUI2 DJSOOL14.I (NOVEL PROTEIN)45..262 185/220 (84%)Se-95 -Homo sapiens (Human), I ..220 197/220 (89%) 220 as (fragment).

Q9CXD6 6230416AOSRIK PROTEIN - Mus musculus (Mouse), 45..262 163/220 (74%) 2e-340 aa. ' 121..340 188/220 (85%) 83 Q9GZT6 MDSO11 (MDS025) (HYPOTHETICAL 29.5 KDA 59..261 96/204 (47%) 6e-PROTEIN) - Homo Sapiens (Human), 254 aa. 50..253 138/204 (67%) 48 PFam analysis predicts that the NOV I3a protein contains the domains shown in the Table 13F.
Table 13F. Domain Analysis of NOVl3a Identities/
Pfam Domain NOVl3a Match Region Similarities Expect Value for the Matched Region Example 14.
The NOV 14 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 14A.

CGTGTTCTTCACCATCTGGATCATTGGAGGAGGCACGACACCCATGTTGTCATGGCTT
AATATCAGAGTTGGTGTTGACCCTGATCAAGATCCACCACCCAACAATGACAGCTTTC
AAGTCTTACAAGGGGACAGCCCAGATTCTGCCAGAGGAAACTGGACAAAACAGGAGAG
CACATGGATATTCAGGCGGTGGTACAGCTTTGATCACAATTACCTGAAGCCCATCCTC
ACACACAGCGGCTCCCCGCTAACCACCACTCTCCCGCCCGCCTGGTGTAGCTTGCTAG
CTCGATGTCTGACCAGTCCCCAGGTGTACGATAACCAAGAGCCACTGAGAGAGGGAAA
CTCTGATTTTATTCTGACTGAAGGCGACCTCACATTGACCTATGGGGACAGCACAGTG
ACTGCAAATGGCTTCTCAGGTTCCCACACTGCCTCCACGAGTCTGGAGGGCAGCTGGA
GAATGAAGAGCAGCTCAGAGGAAGTGCTGGAGCAGGACGTGGGAP.TGGGAAACCAGAA
GGTTTCGAACCAGGGTACCCGCCTAGTGTTTCCTCTGGAAGATAATGTTTGACTTTCC
CTGCAAACCCTGGCACGATGGGGTAGGCTCCCAATGGGGTGAGGATGGCTTCAAGCCC

TAATGTTGCTTGAGGTGGGGCAGTGACTAGATTGAATTAACTCTTCTATTTTATTGGG

GTCTGAAGTTATTGTAACACTTAAAATTTAACTCATGATGCAGATGGTGAGGCAAAAG

TGTCTCTAAATTCAGACAAATGTAGACCTATTTCTACTTTTTTTCACACAGTAGTGCG

CTGTTTCAGAGTTAAACAAACAAAAAAATAGCATACTTTAATGGTCTCTTAATTCATT

CACCTGCAGTGTCTGAACAAGGCAGGGCAGGTGCTGAGTGGGGGGCTTCCTCTTACAA

GAGGCTGCATCTCAGTACACAGTGGTGCAAGTCAAGCTGACCATAGAAATATCAAGTT

AGGGGAGAACAGGCTGGAAGAAAGATTGAGAAGGAAGGCAATTGAGATAGGACCTCCA

AGGATTATGGGAAACTGTTGTTAAATGGAACAGAAAACATGAAAAAATAATATGAGTG

GAGGCTCTGGCAAGGAAGGCTGTGTGACTGCAACCTCATATCAGGATTCCTGACTTTT

ATGCTACCTGTGTTTCTTCTAGACTGA~l.GATTTG_A Z1.A pTnmnTr_Cn TrzLn_CAmTTCAA

CATGAAACAAAGAATTATAGTTCCTTCTCTGGAGATGTCCATAAAGAAGTAATTATGA

TATGTTTAAAACCAGACCGGGTGTGGGGGCTCACGCCTGTAATCCCAGCACTTTGGGA

GGCCGAGGCCGGCGCATCATCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGG

TGAAACCCCGTCTCTACTAP.AAAATACAAATATTAGCCAGGCGTGGTGGCAAGCACCT

TAATCCCAGCTACTTGGGAGGCAGAGGCAGGAGAATCGTTTGAACCCAGGAGGCTGAG

GTTGCAGTGAGCCCAGATAAAGCCACTGCACTCAAACGTGGGGAACAAGAGTGAGACT

TCTCTCAAAAAATAAATAAATAAATAAAATAATATAAAATAAACCAAAGGCAAATAGT

GTTACACTGTTAATTTTTAGTTAATCTGAAGAAAGGAGGTATTTAGAAACTGATGATG

GTATCACTGCAAAAAGCATTAAACTTTTGAGGGCTACCATATGAGCTGACAGCCTAGG

AAATAATTAGTAAGACTGAGGTATCTACTGTGGGTTTAGAAATAACACCAAATTTTGT

AGGATGCTATTATCTGGGGAAGAGAAGGCAGCAAGAAACCTACAAGGCAACAGGCTAG

AAATCAGAGGGAAAGAAATCTCTATGCAAAAAATTAACTGCAAAGAAATACAAGATGG

TAGTCTCAAGACCGGGTAGAATTCTGAACTTTGAGCTGTCGAATGCATGAGATTTCAG

TGGCCACATGAGGAATCAGTGGGAAGTCAATGGAACGTTAAGTATTTTCAACCCACTA

GAAGGTCCTGTCTTCTATAAGTTTAAGAATCTAAGTGTCTTTATGCCATTGAGTGCGG

TGCAGAGAAGGGTCATTTTCCCTTTATCTGGGGAGGCTGCTTCACCAGCCTACCATGT

GGGTGTGATTTGAAAGTTAGGTTTTCAGTTTGGGTTCTTTCTGGATGAGCTGTTCTGT

CCGCCCACACCTGTAGTGCTGAAATACTGAAAGATCTCTCCGGAAAAGTTTGAGTTTC

TCCCCATGTTTCTGTGCTTCAGCAATAGCATTTTTT_TGGCAACCCAATTTCTAAAAA.A

TGCi1TCAATt'1ATGTGGGCATTTTCTTATTATGGCAG~~HlziWt~.W.AUJa;.TA

GTCTACCATAATGAAATCAGCCATTTAATCTTCTCAATTTGCATGTTTAATGGTTAAT

TTTTAAATAAGACATTGCTTCACATCTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGT

CTTGCTCTTTCGCCCGGGCTGGAGTGCAGTCGTGCAACCTCGGCTCGCTGCAATCTCT

GCGCCCCCAGGTTCACGTGATTCTCCTGCCTCAGC_CTCCCTAGTAGCTGGGATTGCAG

GTGCCCACCACCACACCTGGCTAATTTTTTATTTTTAGTAGAGATGGGGTTTTGCCAT

GTTGGCCAGGCTGGTCTTGAACTCCTGACCTTCAGGTGATCCACCTGCCTTGGCCTCC

CGAAGTGCTAGGATTACAGGCATGAGCCACCATGCCCGGCCTGCGTCATAACTTTGTG

TTTGAATTGATAATTTGTGCAAATCAGGAAAATATATATTTAATTAAGTTGAGCCATA

TGAATTTGCTGATATTCAACCATTTTGTAAAAACAGGAGTGGCAATTTCATATGGTTC

AATAAAATAAAATTGAGGCCGGGCACAGTGGCTTACACCCATAATCCCAACACTTTGG

GAGGCTGAAGCAGGAGGATCGCTTGAGCTCAGGAGTTTGAGACCAGACTGAGCAACAT

GGCAGAACTCTGTCTCTACAAAAATACAAAAATGAGACAGGCATGGTGGCACATACCT

TTAGTTCAGTTCTAGGTGATTGGGAGGCTGAGGTGGGAGGATCGCTTGAACCCAAGAG

GCAGAGGATGCAGTGAGCCAAGATCATACCACTGGAAACCAGCCTGGGCAACAGAGTG

AGAACCTGTCTCAAACAAACAAACAAACTGGAATTTATTTTTATGTATGGTATGAGAA

AGGGATTTGTTTTTTTTGTTTTTTTTTTTTTTTTTTTGATATGGAGTCTTACTCTGTT

GCCCAGGCTGGAGTGCAGTGGCGCCATCTTGGCTCACTGCAACTTCTGCCTCCTTTCC

TTTGTTCAAGCGATTCTCTTGTCCCTGAGTAGCTGGGATT_ACAGGCACCGGCCACCAC

GCCCAGCTATTTTTTGTATTTTTAGTAGAGACAGGGTTTC_ACCATGTTGGCCAGGCTG

GTCTCAAACTCCTGACCTCAGGCATTCTGCCCCCCTTGGCCTCCGAAAGTGCTGGGAT

CACAGGCATGAGCCACTGTGCCCAGTCTGAGAAAGGGATTTAATTTACTTTTTTTCTT
CCAAATGGATAGCTCCTTGTCCCAACACTCTCTATTAGTCTGTCATTTCCCAAGTGAT
TTTAAATGTCTCCTTTAACATATACTAAGATTACACACACACACACACACACACACAT
ACACACAAATGGACACATATATGTGTGTGTGTGTATATATATTTTTCTGGACTTTTAA
OIZF Start: ATG at 73 ORF Stop: TGA at 2080 SEQ ID NO: 52 ~ 669 as MW at 73935.6kD
~NOVl4a, MEPGDAALPCPGRVAQAPPRRLLLLLPLLLGRGLRVTAEASASSSGAAVENSSAMEEL

'Protein SequenceLRYGTPGTRGRDKLLNCTQEDQAFSTLVVTFDPEVFFNILLPPVIFHAGYSLKRHFFR

NLGSLLGHSLGTAVSCFRIGNLRYGMVKLMKIMRQLSDKFYYTHCLFFRAIISATDPV

i TVLVIINELHADMDLYVLLFGESILNDVVMWLSSSIVGYQPAGLNTHAFDAAAFLKS

VGIFLGIFSGCFTMGAVTGVVTALVTKFTKLDCFPLLETALFFLMSWSTFLLAEACGF

TGWAVLFCGITQAHYTFNNLSVESRSRSKQLFEAENFIFSCMILALFTFQKHVFSPV

FIIGAFVAVFLGRAAHIYPLSFFLSLGRRHKIGWNFQHTMMFSGLRGAMAFALAICDT

ASYARQMTFPTTPFIVFFTIWIIGGGTTPMLSWLNIRVGVDPDQDPPPNNDSFQVLQG

DSPDSARGNWTKQESTWIFRRWYSFDHNYLKPILTHSGSPLTTTLPPAWCSLLARCLT

SPQVYDNQEPLREGNSDFILTEGDLTLTYGDSTVTANGFSGSHTASTSLEGSWRMKSS

SEEVLEQDVGMGNQKVSNQGTRLVFPLEDNV

Further analysis of the NOV 14a protein yielded the following properties shown in Table 14B.
Table I4B. Protein Sealuence P~-operiies hT~Vi_4a PSort 0.8000 probability located in plasma membrane; 0.4000 probability located in Golgi analysis: body; 0.3000 probability located in endoplasmic reticulum (membrane); 0.0300 probability located in mitochondria) inner membrane SignalP Cleavage site between residues 39 and 40 analysis:
A search of the NOV I 4a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 14C.

Table 14C. Geneseq Results for NOVl4a NOVl4a Identities/

Geneseq Protein/OrganismlLength Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAE16770 Human transporter and ion SS..668 S46/673 (81%)0.0 channel-7 (TRICH-7) protein - Homo 1..672 570/673 (84%) Sapiens, 673 aa. [WO200192304-A2, 06-DEC-2001]

AAB90637 Human secreted protein, 43..517 412/514 (80%)0.0 SEQ ID NO:

.
180 - Homo sapiens, 526 6..519 431/514 (83%) aa.

[W0200121658-Al, 29-MAR-2001]

AAB90SSS Human secreted protein, SS..S 402/502 (80%)0.0 SEQ ID NO: 93 17 - Homo Sapiens, 509 aa. 1..502 420/502 (83%) [W0200121658-Al, 29-MAR-2001]

AAU02883 Human HsNHE-6 polypeptide 13..645 379/641 (S9%)t 0.0 - Homo sapiens, 664 aa. [W0200133945-A1,13..631 450/641 (70%) AAB90591 Human secreted protein, 335..6683021339 (89%)e-174 SEQ ID NO:

129 - Homo Sapiens, 339 1..338 314/339 (92%) aa.

[W0200121658-Al, 29-MAR-2001]
_ . .. _._. ~

In a BLAST search of public sequence datbases, the NOV I4a protein Was found,to have homology to the proteins shown in the BLASTP data in Table 14D.
Table I4D. Public BLASTP Results for NOVl4a Protein NOVl4a Identities/

Accession~ Residues/~ Expect Protein/Organism/Length Similarities for Number Match she lwiaici~edV apnne ResiduesPortion Q96T83 NONSELECTIVE SODIUM 1..668 584/727 (80%)0.0 POTASSIUM/PROTON EXCHANGER 1..724 609/727 (83%) - Homo sapiens (Human), 725 aa.

075827 DJ71L16.S (KIAA0267 LIKE 111..668494/617 (80%)0.0 PUTATIVE NA(+)/H(+) 1..615 517/617 (83%) EXCHANGER) - Homo Sapiens (Human), 616 as (fragment).

Q92581 Sodium/hydrogen exchanger 19..668 414/657 (63%)0.0 (Na(+)/H(+) exchanger 6) 19..654 492/657 (74%) (NHE-6) -Homo sapiens (Human), 669 aa.

Q9U624 SODIUM-HYDROGEN EXCHANGER 52..620 287/654 (43%)e-128 NHE3 - Drosophila melanogaster17..659 378/654 (56%) (Fruit fly), 687 aa.

_-.._......_.........................._.._.~._...._..._................._....._..
..._........_.._... 3 _ . ,~",~"", Q9VM99 NHE3 PROTEIN - Drosophila 52..620 287/654 (43%) e-128 melanogaster (Fruit fly), 727 aa. 57..699 378/654 (S6%) PFam analysis predicts that the NOV 14a protein contains the domains shown in the Table 14E.
Table 14E. Domain Analysis of NOVI4a Identities/
Pfam Domain NOVl4a Match Region Similarities Expect Value for the Matched Region Na_H_Exchanger 75..502 143/472 (30%) 8.I e-103 3 S 11472 (74%) Example 15.
The NOV 1 S clone was analyzed, and the nucleotide and encoded poIypeptide sequences are shown in Table 15A.
Table ISA.
NOV15 Sequence Analysis SEQ ID NO: 53 766 by NOVlSa TATTTGGCGCCCGCTCTCTCTCTCTGTCCCTTTGCCTGCCTCCCTCCCTCCGGATCCC

, ~CTCTCTCCCCGGAGTGGCGCGTCGGGGGCTCCGCCGCTGGCCAGGCGTGATGTTGC

DNA

Sequence ACGTGGAGATGTTGACGCTGGTGTTTCTGGTGCTCTGGATGTGTGTGTTCAGCCAGGA

CCCGGGCTCCAAGGCCGTCGCCGACCGCTACGCTGTCTACTGGAACAGCAGCAACCCC

AGATTCCAGAGGGGTGACTACCATATTGATGTCTGTATCAATGACTACCTGGATGTTT

TCTGCCCTCACTATGAGGACTCCGTCCCAGAAGATAAGACTGAGCGCTATGTCCTCTA

CATGGTGAACTTTGATGGCTACAGTGCCTGCGACCACACTTCCAAAGGGTTCAAGAGA

TGGGAATGTAACCGGCCTCACTCTCCAAATGGACCGCTCAAGTTCvCTvT"~TTCC

AGCTCTTCACTCCCTTTTCTCTAGGATTTGAATTCAGGCCAGGCCGAGAATATTTCTA

CATCTCCTCTGCAATCCCAGATAATGGAAGAAGGTCCTGTCTAAAGCTCAAAGTCTTT

GTGAGACCAACAAATGACACCGTACATGAGTCAGCCGAGCCATCCCGCGGCGAGAACG

CGGCACAAACACCAAGGATACCCAGCCGCCTTTTGGCAATCCTACTGTTCCTCCTGGC

GATGCTTTTGACATTATAGCACAGTCTCCTCCCATCACTTGTCACAGAAAACATCAGG

GTCTTGGAACAC

' ORF Start: ATG at ORF Stop:

at 713 SEQ ID NO: 54 201 as MW at 23295.41eD

NOVISa, MLHVEMLTLVFLVLWMCVFSQDPGSKAVADRYAVYWNSSNPRFQRGDYHIDVCINDYL

CG96S4S-O2 DVF'CPHYEDSVPEDKTERYVLYMVNFDGYSACDHTSKGFKRWECNRPHSPNGPLKFSE

PTOtelri SequenceKFQLFTPFSLGFEFRPGREYFYISSAIPDNGRRSCLKLKVFVRPTNDTVHESAEPSRG

ENAAQTPRIPSRLLAILLFLLAMLLTL

SEQ ID NO: SS 764 by NOVISb TATTTGGCGCCCGCTCTCTCTCTGTCCCTTTGCCTGCCTCCCTCCCTCCGGATCCCCG

, CCCTCTCCCCGGAGTGGCGCGTCGGGGGCTCCGCCGCTGGCCAGGCGTGATGTTGCAC

DNA

Sequence GTGGAGATGTTGACGCTGGTGTTTCTGGTGCTCTGGATGTGTGTGTTCAGCCAGGACC

CGGGCTCCAAGGCCGTCGCCGACCGCTACGCTGTCTACTGGAACAGCAGCAACCCCAG

GTTCCAGAGGGGTGACTACC~P.TATTGATGTCTGTATCAATGACTACCTGGATGTTTTC

TGCCCTCACTATGAGGACTCCGTCCCAGAAGATAAGACTGAGCGCTATGTCCTCTACA

TGGTGAACTTTGGTGGCTACAGTGCCTGCGACCACACTTCCAAAGGGTTCAAGAGATG

GGAATGTAACCGGCCTCACTCTCCAAATGGACCGCTGAAGTTCTCTGAAAAATTCCAG

CTCTTCACTCCCTTTTCTCTAGGATTTGAATTCAGGCCAGGCCGAGAATATTTCTACA

TCTCCTCTGCAATCCCAGATAATGGAAGAAGGTCCTGTCTAAAGCTCAAAGTCTTTGT

GAGACCAACAAATGACACCGTACATGAGTCAGCCGAGCCATCCCGCGGCGAGAACGCG

GCACAAACACCAAGGATACCCAGCCGCCTTTTGGCAATCCTACTGTTCCTCCTGGCGA

TGCTTTTGACATTATAGCACAGTCTCCTCCCATCACTTGTCACAGAAAACATCAGGGT

CTTGGAACAC

ORF Start: ATG at ORF Stop:

at 711 SEQ ID NO: S6 201 as MW at 23237.3kD

NOVlSb, MLHVEMLTLVFLVLWMCVFSQDPGSKAVADRYAVYWNSSNPRFQRGDYHIDVCINDYL

Protein SequenceKFQLFTPFSLGFEFRPGREYFYISSAIPDNGRRSCLKLKVFVRPTNDTVHESAEPSRG

ENAAQTPRIPSRLLAILLFLLAMLLTL

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 1 SB.
Table 15B. Comparison of NOVlSa against NOVlSb.
Protein Sequence NOVlSa Residues/ Identities/
Match Residues Similarities for the Matched Region NOV l Sb 1..186 ' 18S/186 (99%) 1..186 18S/186 (99%) Further analysis of the NOV 1 Sa protein yielded the following properties shown in Table 1 SC.
''able 15C. Protein Sequence Properties NtW' iSa PSort 0.9190 probability located in plasma membrane; 0.2212 probability located in analysis: microbody (peroxisome); 0.2000 probability located in lysosome (membrane); 0.1000 probability located in endoplasmic reticulum (membrane) SignalP Cleavage site between residues 21 and 22 analysis:
S A search of the NOV 15a 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 SD.

Table 15D. Geneseq Results for NOVlSa NOVlSa Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAW00035 HEK4 binding protein - Homo1..201 201/228 (88%)e-115 sapiens, 228 aa. [W09623000-A1, Ol-AUG-1..228 201/228 (88%) 1996]

AAW02586 Lerk-7 protein - Homo Sapiens,1..201 201/228 (88%)e-115 228 aa.

[W09617925-A1, 13-JLTN-1996]1..228 201/228 (88%) AAR97854 Human AL-1, a ligand for 1..201 201/228 (88%)e-115 eph-related "

tyrosine kinase receptor 1..228 201/228 (88%) REK7 - Homo Sapiens, 228 aa. [W09613518-A1, MAY-1996]

ABG27837 Novel human diagnostic protein4..201 i 98/225 ' e-113 #27828 - (88%) Homo Sapiens, 335 aa. [W0200175067-111..335198/225 (88%) A2, I1-OCT-2001]

ABG27837 Novel human diagnostic protein4..201 198/225 (88%)e-113 #27828 -Homo sapiens, 335 aa. [W0200175067-1 11..335198/225 (88%'0) A2, 11-OCT-2001]

In a BLAST search of public sequence datbases, the NOV 15a protein was found to have homology to the proteins shown in the BLASTP data in Table 15E.
Table 15E. Public BLASTP Results for NOVlSa Protein NOVISa Identities/

AccessionProtein/OrganismlLength Residues/SimilaritiesExpect for Number . _ Match s the MatchesValue ~

ResiduesPortion P52803 Ephrin-AS precursor (EPH-related1..201 20I/228 (88%)e-115 receptor tyrosine kinase 1..228 201/228 (88%) ligand 7) CLERK-7) (AL-1 ) - Homo sapiens (Human), 228 as.

P97605 Ephrin-AS precursor (EPH-related1..201 199/228 (87%)e-114 receptor tyrosine kinase 1..228 200/228 (87%) ligand 7) CLERK-7) (AL-1 ) - Rattus norvegicus (Rat), 228 as.

008543 Ephrin-AS precursor (EPH-related1..201 199/228 (87%)e-114 receptor tyrosine kinase 1..228 200/228 (87%) ligand 7) CLERK-7) (AL-1 ) - Mus musculus (Mouse), 228 as.

P52804 Ephrin-AS precursor (EPH-related1..201 181/28 (79%)e-102 1..228 186/228 (81%) 7) (RAGS protein) - Gallus gallus (Chicken), 228 aa.
P79728 Ephrin-AS precursor (EPH-related 1..201 152/229 (66%) 3e-85 receptor tyrosine kinase ligand 7) CLERK- 1..228 173/229 (75%) 7) (AL-1) (ZFEPHL4) - Brachydanio rerio (Zebrafish) (Zebra danio), 228 aa.
PFam analysis predicts that the NOV 1 Sa protein contains the domains shown in the Table 1 SF.
Table 15F. Domain Analysis of NOVlSa Identities!
Pfam Domain NOVlSa Match Region Similarities ~ Ezpect Value for the Matched Region Ephrin 26..164 86/148 (58%) 7.8e-91 138/148 (93%) Egamule 16.
The NOV 16 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 16A.
Table 16A.~NOVl6 Sequence Analysis SEQ ID NO: 57 ~ ~~ 369 bp~
~~~

NOVl6a, CCCAGGTGAAGATCTGGAGGCACTCCTATGATGTCCCACCACCTCCAATGGAGCCCAA

DNA

Sequence GCCCCTCCTCAGAGCAGCTGCCGCAGCCCGAGGTAATCTCGGCCCCGCGCCGGGGCTG

GCTGGGCAGCACCCGAC,~~,C
; ;r,TmnTCAG ;ATVGCAGGTVAGAGATGGCAAiiC-rG

CAGGCACCGCAGGGGCGAATCAGGTAGGCTACCCCAGCCAGGATTGTTCTTGTACAAG

TGTTTGTATGACCAGATGCTTTCAGTTCTCTTGAACATATACCTAGAAGTAGAATTTC

TGGGTCATATGGTAATTTTAT

ORF Start: ATG at ORF
28 Stop:
TGA
at SEQ ID NO: 58 98 as MW at 10060.2kD

NOVl6a, MMSHHLQWSPTAGGLGEEGAGQWTGGSEGRPLLRAAAAARGNLGPAPGLAGQHPSTGI

CG97101-O1 I~AGQRWQTAGTAGANQVGYPSQDCSCTSVCMTRCFQFS

Protein Sequence Further analysis of the NOV 16a protein yielded the following properties shown in Table 16B.

Table 16B. Protein Sequence Properties NGVl6a PSort 0.8061 probability located in lysosome (lumen); 0.6027 probability located in analysis: microbody (peroxisome); 0.4500 probability located in cytoplasm;
0.1000 probability located in mitochondria) matrix space SignalP No Known Signal Sequence Predicted analysis:
A search of the NOV 16a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 16C.
Table 16C. Geneseq Results for NOVl6a NOVl6a ~ Identities/t Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value Residues Region AAUI Spider natural silk protein13..79 26167 (38%) 0.011 1781 Spidroin 1 -Nephila clavipes, 651 aa. 90..151 29/67 (42%) [W0200190389-A2, 29-NOV-2001]

AAY59070N. clavipes spider silk 13..79 26/67 (38%) 0.011 protein 1 -Nephila clavipes, 718 aa. 90..151 29/67 (42%) [LJS5989894-A, 23-NOV-1999]

AAY40097Spider silk protein spidroine13..79 26/67 (38%) 0.01 major 1 - I

Nephila clavipes, 651 aa. 90..151 29/67 (42%) [FR2774588-A1, 13-AUG-1999]

AAW53346Nephila clavipes spider 13..79 26/67 (38%) 0.011 silk protein -Nephila clavipes, 718 aa. 90..151 29/67 (42%) [US5728810-A, 17-MAR-1998]

AAR14308N.clavipes dragline silk 13.:79 26/67 (38%) 0.011 protein-1 - ~

Nephilia clavipes, 718 aa. 90..151 29/67 (42%) [EP452925-A, In a search of public sequence NOV 16a BLAST datbases, the protein was found to have S homology to the proteins shown in the BLASTP data in Table 16D.

Table 16D. Public BLASTP Results for NOVl6a Protein NOVl6a Identities/

AccessionProtein/Organism/Length . Residues/SimilaritiesExpect for Number Match the MatchedValue ResiduesPortion Q9UGU4 DJ526I14.1 (PERIPHERAL 2..94 48/94 (51%)4e-18 BENZODIAZEPINE RECEPTOR 1..94 54/94 (57%) RELATED PROTEIN (ISOFORM 2)) -Homo sapiens (Human), 102 aa.

Q13849 PERIPHERAL BENZODIAZEPINE 2,.94 47/94 (50%)3e-17 RECEPTOR RELATED PROTEIN - 1..94 53/94 (56%) Homo sapiens (Human), 102 aa.

A36068 major ampullate fibroin protein' 13..7926/67 (38%)' 0.025 - orb spider ' (Nephila clavipes), 718 as 90..151 29/67 (42%) (fragment).

Q8WSW4 DRAGLINE SILK PROTEIN -Nephila13..79 31/67 (46%)0.025 clavipes (Orb spider), 644 47..104 35/67 (51%) as (fragment).

046172 DRAGLINE SILK PROTEIN SPIDROIN13..79 31/67 (46%)0.025 ~

1 - Nephila clavipes (Orb 44..1 35/67 (51 spider), 617 as Ol %) (fragment).

PFam analysis predicts that the NOV 16a protein contains the domains shown in the Table I6E.
Table 16E. Domain Analysis of NOVl6a Identities/
Pfam Domain NOVl6a Match Region Similarities ~ ~~_E_ xoect value for the Matched Region Example 17.
The NOV 17 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 17A.

WO 02/090504 PCT/US02/14342 ..
TACTATATTATAGTATTTGTTTTCATTTTTTTGTTAAGTTCCATCTTGAACGGATTAA

GTATATATCTATTAACTAGAGGCGAAAATATAATTTATTCTCTAACTAACAAAGTTTG

GAATCATATTTTAAGATTAAAAACGTCTTTTTTTGATAAAAATAGTAATGGTGAACTA

TTAAGTAGAATTATAGATGACACTAAATCAATAAACAGTTTCATTACAGAAATTATAC

CATCTTTTTTTCCATCAATAATTGTACTATTTGGATCAATCTTTTTTTTATTTATGCT

AGATTGGGAAACAGCTTTAATTGCTCTTATTTCAATACCTATGTATGTCATTTTAATA

ATACCAATAAGCAACGTAATGCAAAAACTTTCTTATAAAACACAACTTGAAACTGCTA

AGGTTAGTGGTGTAATAGCTCATGTTTTATCTAAAATCAAATTAGTTAAACTTTCAAA

TTCAATTAATAAAGAGTTTCGTCAAACTAATTCATATTTACGAAATATATATTATTTG

GGGGTGAAAGAAGGTGTTATCAATTCAATTGTAGTACCTCTTTCTACGTTAATTATGC

TTGTTTCAATGGGGGGTGTATTAGGTTTTGGAGGATATAGAGTGGCATCTGGAGCCAT

ATCTCCTGGCACGCTTATTGCTCTTATTTTTTATATGACTCAATTAACTGACCCTATT

GAAAAP,ATATCTAGTCTCTTTACAGGATATAAAAAAACTATAGGTGCAAGTCAAAGAC

TTTCTGAAATATTAAGTGAAGAAAAAGAAAATTTACAAAATAATAATCTTAATATTTT

AAATTCAGTAGATTTATCATTTAATAACGTATCTTTCAGTTATGATGAAAACAATCAT

GTTTTTACTAATTTATCCTTTACTATACCTAAAAATAAAATAACTGCTATAGTAGGTC

CTTCCGGTTCTGGTAAAACAACTATTCTTAATTTGATTTCAAGACTATATGAAATTCA

AAGTGGTTCAATTAAGTATGGAACTAATTCTATTTATGACTATTCTTTAGTTAATTGG

AGAAAAAATTTAGGATATGTTATGCAAAACTCTGGTGTATTGAATAGAACAGTC.~AAAA

GCAATATTACTTATTCGCTACAAGAGACACCATGTATAGAAGATATCATTTATTATTC

TAAGCTAGCGTC_n n rGCamr_ammmmAm~pTGp~AT~p~CCTAF~TGP'I'T~~~
~TnCGC-,.., ATTGGAGAAAAAGGAATTAATTTATCTGGCGGTGAAAAACAAAGGTTAGATATAGCTA

GAAACTTTATTAAAACACCTGGGATTTTGTTGTTAGATGAAGCTACTTCAAATTTAGA

TAGCGAAAGTGAACACAAAATACAAGAATCTATAAAAAATGTTAGCAACGATAGAACA

ACAATAATAGTAGCGCATCGTCTTTCCACTGTACTAAAAGCTGATAAAATAATTTTTA

TCGATAATGGTGAAATTACAGGAATGGGTACTCATGAAGAGTTATTAGCTAGACATTC

AAAATATAAAAATATGATTGAGCTACAACAATTAAAGTAAGATATTCAGAATTATATG

ACATATACT

OIRF Start: ATG at 7 ORF
Stop: TAA at 1720 SEQ ID NO: 60 571 as MW at 64349.O1eD

NOVl7a, MKQKNPVLHLVNEIEIPKWLLFFSVLLSIIGSTFQLIVPLFTQNIVDNFSEVIKNKYY

Protein SequenceRIIDDTKSINSFITEIIPSFFPSIIVLFGSIFFLFMLDWETALIALISIPMYVILIIP

ISNVMQKLSYKTQLETAKVSGVIAHVLSKIKLVKLSNSINKEFRQTNSYLRNIYYLGV

KEGVINSIWPLSTLIMLVSMGGVLGFGGYRVASGAISPGTLIALIFYMTQLTDPIEK

ISSLFTGYKKTIGASQRLSEILSEEKENLQNNNLNILNSVDLSFNNVSFSYDENNHVF

TNLSFTIPKNKITAIVGPSGSGKTTILNLISRLYEIQSGSIKYGTNSIYDYSLVNWRK

NLGYVMQNSGVLNRTVKSNITYSLQETPCIEDIIYYSKLASTHDFIMKLPNDYNTLIG

EKGINLSGGEKQRLDIARNFIKTPGILLLDEATSNLDSESEHKIQESIKNVSNDRTTI

IVAHI2LSTVLKADKi I FIiu7Gr;1 T'GInGT"~~Li~H$T~Y ~.~'1'NiiELSlc,~Ln Further analysis of the NOV 17a protein yielded the following properties shown in Table 17B.
Table 17B. Protein Sequence Properties NOVl7a PSort 0.6400 probability located in plasma membrane; 0.4600 probability located in Golgi analysis: body; 0.3700 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen) SignalP Cleavage site between residues 36 and 37 analysis:

A search of the NOV 17a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 17C.
Table I7C. Geneseq Results for NOVl7a NOVl7a Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect [Patent #, for IdentifierDate) Match the Matched Value ResiduesRegion ABB48756Listeria monocytogenes protein19..571 200/554 (36%)e-116 #1460 -Listeria monocytogenes, 23..575 349/554 (62%) 575 aa. ~

[W0200177335-A2, 18-OCT-2001 ]

AAU36908Staphylococcus aweus cellular~ 18..570~ 180/569 ~ 7e-81 (31%) proliferation protein #107813-.~78 3n9/569153~f1 -Staphylococcus aureus, 578 aa.

[W0200170955-A2, 27-SEP-2001]1 AAU36908Staphylococcus aureus cellular18..570 180/569 (31%)7e-81 ~

proliferation protein #107813..578 309/569 (53%) -Staphylococcus aureus, 578 aa.

[W0200170955-A2, 27-SEP-2001i ]

AAE02437Human ATP binding cassette,29..570 172/557 (30%)~ I
ABCB9 e-76 transporter protein - Homo ; 196..743296/557 (52%) sapiens, 766 aa. [W0200140305-AI, 07-JUN-2001]

AAG79246Amino acid sequence'of a i 29..570172/557 (30%)1e-76 human TAP-Like (HUTAPL) polypeptide 196..743296/557 (52%) - Homo sapiens, 766 aa. [W0200173018-A2, In a BLAST search of public sequence datbases, the NOV 17a protein was found to have S . - - >romology to the proteins shown in the BLASTP data it T able 17 D.
Table 17D. Public BLASTP
Results for NOVI7a Protein NOVl7a Identities/

AccessionProtein/Organism/Length Residues/SimilaritiesExpect for Number Match the Matched Yalue ResiduesPortion Q93GF4 AURT - Staphylococcus aureus,I..570 283/572 (49%)e-164 571 aa.

1..571 414/572 (71%) Q99VX4 ATP-BINDING CASSETTE 1..570 2731574 (47%)e-160 TRANSPORTER A - Staphylococcus1..573 413/574 (71 %) aureus (strain Mu50 / ATCC
700699), and, 575 aa.

P72354 ATP-BINDING CASSETTE 1..570 272/574 (47%)e-159 I..573 413/574 (71%) aureus, 575 aa. ~ ~ ~~,~~~-~~-~~~

Q54121 PEPT - Staphylococcus epidermidis,278/571 (48%) e-157 571 1..570 aa. 1..570 401/571 (69%) Q53614 ABCA - Staphylococcus aureus, 268/574 (46%) e-156 575 aa. 1..570 1..573 407/574 (70%) PFam analysis predicts that the NOV 17a protein contains the domains shown in the Table 17E.
Table 17E. Domain Analysis of NOVl7a Identities/

Pfam Domain NOVl7a Match RegionSimilarities Expect Value for the Matched Region transmembrane4~ 18..69 17/52 (33%) 0.078 40/52 (77%) ABC_membrane21..288 82/285 (29%) 3.4e-32 187/285 (66%) PRK ~ 360..378 8/19 (42%) 0.22 - ~ I 4/ 19 (74%) ABC_tran 358..543 61/199 (31%) 8.1e-49 145/199 (73%) Example 18.
The NOV 18 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 18A.
Table 18A. NOV18 Sequence Analysis ~ SEQ ID NO: 61 ( 103 8 by NOVIBa, CAGGCACCGGCGTTAGCGGGTCGCCGACCCGCAATCCCCGCC

Sequence CCGCTCTGGCGGCCAGGCCGAGAGGGACAGAGACTGGAGCCA
CGAGCACGTCCCGCGGGCCGGGCGGCTCGCAGGGGTCGCAGGGCCCCTCGCCTCAGGG
CTTGAACCCAAGAGCAACACTTACATCCTCATCAACACCCTGGAGCCTCCTGTGGAGG
CTTAGGGCTCATCTTTATGAAGGGCAACACCATCAAGGAAACTGAAGCCTGGGACTTT
CTGCGGCGCTTAGGGGTCTACCCCACCAAGAAGCATTTAATTTTCGGAGATCCAAAGA

AAGATGAAAGTTCTTAAGTTTGTGGCCAAGGTCCATAATCAAGACCCCAAGGACTGGC

CAGCGCAGTACTGTGAGGCTTTGGCAGATGAGGAGAACAGGGCCAGACCTCAGCCTAG

TGGCCCAGCTCCATCCTCTTGAAAGGTGGATTCAGAGGGACCCCCGGGACAA

ORF Start: ATG at ORF Stop:

at 1006 SEQ ID NO: 62 305 as MW at 34404.8kD

NOVl8a, MLQKPRNRGRSGGQAERDRDWSHSGNPGASRAGEDARVLRDGFAEEAPSTSRGPGGSQ

Protein SequenceGDYKDTFPDLFKRAAERLQYVFGYKLVELEPKSNTYILINTLEPPVEEDAEMRGDQGT

PTTGLLMIVLGLIFMKGNTIKETEAWDFLRRLGVYPTKKHLIFGDPKKLITEDFVRQR

YLEYRRIPHTDPVDYEFQWGPRTNLETSKMKVLKFVAKVWQDPKDWPAQYCEALADE

ENRARPQPSGPAPSS

SEQ ID NO: 63 727 by NOVI8b, AGACATGTTGCAAAAACCGAGGAACCGGGGCCGCTCTGGCGGCCAGGCCGAGAGGGAC

DNA

Sequence TTCTCAGAGACGGCTTTGCCGACATACTGAAGCACGTCATCGGGGACTACAAGGACAT

CTTCCCCGACCTCTTCAAACGGGCCGCCGAGCGCCTCCAGTACGTCTTCGGGTATAAG

CTGGTGGAACTTGAACCCAAGAGCAACACTTACATCCTCATCAACACCCTGGAGCCTG

TGGAGGAGGATGCCGAGATGAGGGGTGACCAAGGCACGCCCACTACGGGCCTCCTGAT

GATCGTCTTAGGGCTCATCTTTATGAAGGGCAACACCArCAAGGAAACTGAAGCCTGG

GACTTTCTGCGGCGCTTAGGGGTCTACCCCACCAAGAAGCATTTAATTTTCGGAGATC

CAAAGAAACTCATTACTGAGGACTTTGTGCGACAGCGTTACCTGGAATACCGGCGGAT

ACCCCACACCGACCCCGTCGACTACGAATTCCAGTGGGGCCCGCGAACCAACCTGGAA

ACCAGCAAGATGAAAGTTCTTAAGTTTGTGGCCAAGGTCCATAATCAAGACCCCAAGG

ACTGGCCAGCGCAGTACTGTGAGGCTTTGGCAGATGAGGAGAACAGGGCCAGACCTCA

GCCTAGTGGCCCAGCTCCATCCTCTTGAAAG

ORF Start: ATG at ORF' 5 Stop:
TGA
at 722 SEQ ID NO: 64 239 as MW at 27463.9kD

NOVlBb, MLQKPRNRGRSGGQAERDRDWSHSGNPGASRAGEDAR.VI~RDGFADILKHVIGDYKDIF

Protein Sequence~GLIFMKGNTIKETEAWDFLRRLGVYPTKKHLIFGDPKKLITEDFVRQRYLEYRRIP

HTDPVDYEFQWGPRTNLETSKMKVLKFVAKVHNQDPKDWPAQYCEALADEENRARPQP

SGPAPSS

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 18B.
Table 188. Comparison of NOVl8a against NOVl8b.
Protein Sequence NOVl8a Residues/ Identities/
Match Residues Similarities for the Matched Region NOVl8b 109..305 196/197 (99%) 44..239 196/197 (99%) Further analysis of the NOV 18a protein yielded the following properties shown in Table 18C.

Table 18C. Protein Sequence Properties NOVlBa PSort 0.4500 probability located in cytoplasm; 0.1000 probability located in mitochondria) analysis: matrix space; 0.1000 probability located in lysosome (lumen); 0.0806 probability located in microbody (peroxisome) SignalP No Known Signal Sequence Predicted ~alysis:
A search of the NOV 18a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 18D.
Table 18D. Geneseq Results for NOVl8a f NOVl8aIdentities/ 1 Geneseq Protein/Organism/Length Residues/Similarities Expect [Patent #, for IdentifierDatej Match the Matched Value ResiduesRegion ABB 11541Human melanoma Ag homologue,90..305 213/216 (98%)e-122 ~SEQ

ID N0:1911 - Homo Sapiens, 1..215 213/216 (98%) 215 aa.

[W0200157188-A2, 09-AUG-2001]

AAB60476 Human cell cycle and proliferationI..302 151/304 (49%)1e-79 protein CCYPR-24, SEQ ID 1..293 203/304 (66%) N0:24 -Homo Sapiens, 308 aa. [W0200107471-A2, O 1-FEB-2001 ]

AAY79141 Human haemopoietic stem 46..287 109/243 (44%)1 e-55 cell regulatory protein SCMI 13 - Homo Sapiens,240..479160/243 (64!) 606 aa.

[W0200008145-A2, 17-FEB-2000]

AAB94174 Human protein sequence SEQ 79..294 102/217 (47%)2e-52 ID

NO:14482 - Homo Sapiens, 438..650150/217 (69%) 706 aa.

[EPI074617-A2, 07-FEB-2001]

AAB92822 Human protein sequence SEQ 79..294 102/217 (47%)3e-52 ID

NO:11353 - Homo sapiens, 297..509150/217 (69%) 565 aa.

[EP I 074617-A2, 07-FEB-2001 ]

In a BLAST search of public sequence datbases, the NOV 18a protein was found to have homology to the proteins shown in the BLASTP data in Table I 8E.

Table 18E. Public BLASTP
Results for NOVl8a Protein NOVl8a Identities/

AccessionProtein/OrganismlLength Residues/SimilaritiesExpect for Number Match the MatchedValue ResiduesPortion Q96MG7 CDNA FLJ32395 FIS, CLONE 1..305 304/305 e-177 (99%) SKMUS2000117, MODERATELY 1..304 304/305 (99%) SIMILAR TO HOMO SAPIENS

MAGEF 1 MRNA - Homo Sapiens (Human), 304 aa.

Q9CPR8 5730494G16RIK PROTEIN IMAGE-G1)1..305 233/306 e-128 (76%) - Mus musculus (Mouse), 279 1..279 250/306 aa. (81%) Q9D378 5730494G16RIK PROTEIN - Mus ' 1..3 ' 2321306 i e-i VJ ( 75 io) 2 i musculus (Mouse), 279 aa. 1..279 249/306 (80%) BAB84964 FLJ00211 PROTEIN - Homo Sapiens85..290 205/206 e-116 (99%) (Human), 213 as (fragment). 1..205 205/206 (99%) Q99PB1 MAGE-G2 -Mus musculus (Mouse),1..305 201/306 e-106 294 (65%) aa. 9..294 226/306 (73%) PFam analysis predicts that the NOV I 8a protein contains the domain shown in the Table 18F.
Table 18F. Domain Analysis of NOVl8a Identities/
Pfam Domain NOVl8a Match Region Similarities Expect Value for the Matched Region ~
MAGE 1..209 781262 (30%) 1.5e-33 144/262 (55%) Example 19.
The NOV 19 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 19A.

GCTGAACCTGTGTGAGGATGGTCCATGTCACAAACGGCGGGCAAGCATCTGCTGTACC
CAGCTGGGGTCCCTGTCGGCCCTGAAGCATGCTGTCCTGGGGCTCTACCTGCTGGTCT
CGACCTGAAGGCCCTGACTCGCAATGTGAACCGGCTGAATGAGAGCTTCCGGGACTTG
GTGGCCCTGCTGCGGGACCGCACGGGCCAGCAGAGCGACACGGCGCAGCTGGAGCTCT
GCTGGACGGGCTGGCGCGCAGGGTGGGCATCCTGGGCGAGGAGCTGGCCGACGTGGGC
CCCTCGCGAAAGGGCCCCCGGGACCCAAAGGTGA
CAGAGCTGGGGATGCCAGTGGCGTGGAGGCCCCGATGATGATCCGCCTGGTGAATGGC
TCAGGTCCGCACGAGGGCCGCGTGGAAGTGTACCACGACCGGCGCTGGGGCACCGTGT
CGGTGTGGAGGAGGTGTACCGCACAGCTCGATTCGGGCAAGGCACTGGGAGGATCTGG
CTGAAAGTGGGCAGA
ORF Start: ATG at 17 ORF Stop: TGAat 1568 SEQ ID NO: 66 517 aa~~MW at 56256.2kD
NOVl9a, MLRSEGTRLYSLSQGHSAVAGCDGEQTMYLHTVSDCDTSSICEDSFDGRSLSKLNLCE

Protein Sequence TLNESFRDLQLRLLQAPLQADLTEQVWKVQDALQNQSDSLLALAGAVQRLEGA
LWGLQAQAVQTEQAVALLRDRTGQQSDTAQLELYQLQVESNSSQLLLRRHAGLLDGLA
VTEDLRLKDWEHSIALRNISLAKGPPGPKGDQGDEGKEGRPGIP
SGVEAPMMIRLVNGSGPHEGRVEVYHDRRWGTVCDDGWDKKDGDWCRMLGFR~
YRTARFGQGTGRIWMDDVACKGTEETIFRCSFSKWGVTNCGHAEDASWCNRH
SEQ ID NO: 67 903 by NOVl9b, CCACCATGGGAAATGCTGAGATCTGAGGGGACAAGGCTCTACAGCCTCAGCCAGGCGC
CCn'~~~C l'~ DNA
~p'CTCAGCTGTTGCAGGGTGTGirivGAVT~CAi~c~i~i:TATGTACCTACACACCGTc:iW CG
J l'TJ -V~.
Sequence ACTGTGACACCAGCCCCATCTGTGAGGATTCCTTTGATGGCAGGAGCCTGTCCAAGCT
GAACCTGTGTGAGGATGGTCCATGTCACAAACGGCGGGCAAGCATCTGCTGTACCCAG
CTGGGGTCCCTGTCGGCCCTGAAGCATGCTGTCCTGGGGCTCTACCTGCTGGTCTTCC
TGATTCTTGTGGGCATCTTCATCTTAGCAGGGCCACCGGGACCCAAAGGTGATCAGGG
GGATGAAGGAAAGGAAGGCAGGCCTGGCATCCCTGGATTGCCTGGACTTCGAGGTCTG
CCCGGGGAGAGAGGTACCCCAGGATTGCCCGGGCCCAAGGGCGATGATGGGAAGCTGG
GGCGCTGGGGCACCGTGTGTGACGACGGCTGGGACAAGAAGGACGGAGACGTGGTGTG
CCGCATGCTCGGCTTCCGCGGTGTGGAGGAGGTGTACCGCACAGCTCGATTCGGGCAA
GGCACTGGGAGGATCTGGATGGATGACGTTGCCTGCAAGGGCACAGAGGAAACCATCT
TCCGCTGCAGCTTCTCCAAATGGGGGGTGACAAACTGTGGACATGCCGAAGATGCCAG
CGTGACATGCAACAGACACTGAAAGTGGGCAGA
Start: ATG at 80 ORF Stop: TGA at 890 SEQ ID NO: 68 270 as ~MW at 28880.SkD
NOVl9b, ~MENFtAMXYLHTVSDCDTSPICEDSFDGRSLSKLNLCEDGPCHKRRASICCTQLGSLSAL

Protein Sequence ~GLPGPKGDDGKLGATGPMGMRGFKGDRGPKGEKGEKGDRAGDASGVEAPMMIRLVNGS
DDVACKGTEETIFRCSFSKWGVTNCGHAEDASVTCNRH
Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 19B.
Table 19B. Comparison of NOVl9a against NOVl9b.
Protein Sequence NOVl9a Residues/ Identities/
Match Residues Similarities for the Matched Region NOVl9b 329..517 141/189 (74%) 82..270 141/189 (74%) Further analysis of the NOV 19a protein yielded the following properties shown in Table 19C.
Table 19C. Protein Sequence Properties NOVl9a PSort 0.8000 probability located in mitochondria) inner membrane; 0.6500 probability analysis: located in plasma membrane; 0.3000 probability located in microbody (peroxisome);
0.3000 probability located in Golgi body SignalP ~ No Known Signal Sequence Predicted analysis:
A search of the NOV 19a protein against the Geneseq database, a proprietary database~that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 19D.
Table 19D. Geneseq Results for NOVl9a NOVl9a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate) Match the Matched Value ResiduesRegion AAE08824Human scavenger receptor 98..517 399/420 (95%)0.0 like protein -Homo Sapiens, 435 aa. [W0200157260-16..435 399/420 (95%) A1, 09-AUG-2001]

AAE08846Human scavenger receptor 105..517394/413 (95%)0.0 like protein mature sequence - Homo Sapiens,1..413 394/413 (95%) aa. [W0200157260-Al, 09-AUG-2001]

AAE08823Human partial scavenger 329..455126/127 (99%)6e-75 receptor like protein - Homo Sapiens, 1..127 127/127 (99%) 127 aa.

[W0200157260-A1, 09-AUG-2001]

AAW 19708Macrophage scavenger receptor protein164/500 (32%)I e-69 - 25..514 Homo Sapiens, 451 aa. [US5624904-A,247/500 (48%) 2..449 29-APR-1997]

AAR27036 Bovine sol. scavanger receptor - 161/482 (33%)1 e-68 Bos 37..514 taurus, 453 aa. [W09214482-A, 03-SEP-249/482 (51%) 13..451 1992) In a BLAST search of public sequence datbases, the NOV 19a protein was found to have homology to the proteins shown in the BLASTP data in Table 19E.
Table 19E. Public BLASTP
Results for NOVl9a Protein NOVl9a Identities/

AccessionProtein/Organism/Length ~ SimilaritiesExpect Residues/for Number Match she i~~aichedV aiue i ResiduesPortion Q91 WD6 SIMILAR TO RIKEN CDNA 26..514 430/489 (87%)0.0 4933425F03 GENE - Mus musculus4..488 449/489 (90%) (Mouse), 491 aa.

Q9CUC3 4933425F03RIK PROTEIN - Mus 26..397 329/372 (88%)0.0 musculus (Mouse), 375 as 4..375 344/372 (92%) (fragment).

Q9D4G8 4932433F15RIK PROTEIN - Mus 130..403243/274 (88%)e-139 musculus (Mouse), 280 aa. 1..274 258/274 (93%) P21758 Macrophage scavenger receptor37..514 166/482 (34%)1 e-70 types I

and II (Macrophage acetylated13..451 249/482 (51%) LDL

receptor I and II) - Bos taurvs (Bovine), 453 aa.

Q05585 Macrophage scavenger receptor77..514 161/450 (35%)3e-70 types I

and II (Macrophage acetylated46..452 236/450 (51%) LDL

receptor I and II) - Oryctolagus cuniculus (Rabbit), 454 aa.

PFam analysis predicts that the NOV 19a protein contains the domains shown in the Table 19F.
Table 19F. Domain Analysis of NOVl9a Identities/
Pfam Domain NOVl9a Match Region Similarities Expect Value for the Matched Region Collagen 337..396 28/60 (47%) 9.1e-13 43/60 (72%) SRCR 418..515 ~ 44/114 (39%) 2e-33 81/114 (71%) Example 20.
The NOV20 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 20A.
Table 20A. NOV20 Sequence Analysis SEQ ID NO: 69 ~ 1538 by NOV20a, TGGCGCCCAGCGGGGTCATGGTGCCCGGCGCCCGCGGCGGCGGCGCACTGGCGCGGGC

Sequence GCGGAGGAGCTGGGTGATGGCTGTGGACACCTAGTGACTTATCAGGATAGTGGCACAA
TGACATCTAAGAATTATCCCGGGACCTACCCCAATCACACTGTTTGCGAAAAGACAAT
CAGACCTGTGCTTCTGACTATCTTCTCTTCACCAGCTCTTCAGATCAATATGGTCCAT
ACTGTGGAAGTATGACTGTTCCCAAAGAACTCTTGTTGAACACAAGTGAAGTAACCGT
CCGCTTTGAGAGTGGATCCCACATTTCTGGCCGGGGTTTTTTGCTGACCTATGCGAGC
TATGGTAGATGGATATAGAGATACCTCTTTAfiTGTGCAAAGCTGCCATCCATGCAGGA
ATAATTGCTGATGAACTAGGTGGCCAGATCAGTGTGCTTCAGCGCAAAGGGATCAGTC
CATTACAACGGfiGGCTATTCCATTGGTGCTCCTTGTTGTCCTGGTGTTTGCTGGAATG
GGGATCTTTGCAGCCTTTAGAAAGAAGAAGAAGAAAGGAAGTCCGTATGGATCAGCAG
AGGCTCAGAAAACAGACTGTTGGAAGCAGATTAAATATCCCTTTGCCAGACATCAGTC
AGCTGAGTTTACCATCAGCTATGATAATGAGAAGGAGATGACACAAAAGTTAGATCTC
ATCACAAGTGATATGGCAGATTACCAGCAGCCCCTCATGATTGGCACCGGGACAGTCA
CGAGGAAGGGCTCCACCTTCCGGCCCATGGACACGGATGCCGAGGAGGCAGGGGTGAG
CTGCCCCTGGCGCCCCCGGAGCCCGAGTACGCCACGCCCATCGTGGAGCGGCACGTGC
Start: ATG at ? 8 1 ORS' Stap: TG a at 1 s36 ID NO: 70 1506 as BMW at 54247.6kD
Sequence VPKELLLNTSEVTVRFESGSHISGRGFLLTYASSDHPDLITCLERASHYLKTEYSKFC
PAGCRDVAGDISGNMVDGYRDTSLLCKAAIHAGIIADELGGQISVLQRKGISRYEGIL
ANGVLSRESSLGINITTVAIPLVLLVVLVFAGMGIFAAFRKKKKKGSPYGSAEAQKTD
CWKQIKYPFARHQSAEFTISYDNEKEMTQKLDLITSDMADYQQPLMIGTGTVTRKGST
FRPMDTDAEEAGVSTDAGGHYDCPQRAGRHEYALPLAPPEPEYATPIVERHVLRAHTF
SAQSGYRVPGPQPGHHIiSLSSGGFSPVAGVGAQDGDYQRPHSAQPADRGYDRPKAVSA
SEQ ID NO: 71 X636 by 199652779 DNA ~fiGACATCTAAGAATTATCCCGGGACCTACCCCAATCACACTGTTTGCGAAAAGAC
Sequence ~~TTACAGTACCAAAGGGGAAAAGACTGATTCTGAGGTTGGGAGATTTGGATATCGAA
CATACTGTGGAAGTATGACTGTTCCCAAAGAACTCTTGTTGAACACAAGTGAAGTAAC
CGTCCGCTTTGAGAGTGGATCCCACATTTCTGGCCGGGGTTTTTTGCTGACCTATGCG

CAGAATACAGCAAATTCTGCCCAGCTGGTTGTAGAGACGTAGCAGGAGACATTTCTGG

GAATATGGTAGATGGATATAGAGATACCTCTTTATTGTGCAAAGCTGCCATCCATGCA

GGAATAATTGCTGATGAACTAGGTGGCCAGATCAGTGTGCTTCAGCGCAAAGGGATCA

GTCGATATGAAGGGATTCTGGCCAATGGTGTTCTTTCGAGGGAGTCTTCTCTCGAG

ORF Start: at 1 ORF Stop:
end of sequence SEQ ID NO: 72 212 as MW at 22920.41cD

NOV20b, GTEELGDGCGHLVTYQDSGTMTSKNYPGTYPNHTVCEKTITVPKGKRLILRLGDLDIE

Protein Sequence SSDHPDLITCLERASHYLKTEYSKFCPAGCRDVAGDISGNMVDGYRDTSLLCKAAIHA

GIIADELGGQISVLQRKGISRYEGILANGVLSRESSLE

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 20B.
Table 20B. Comparison of NOV20a against NOV20b.
Protein Sequence NOV20a Residues," Identities/
Match Residues Similarities for the Matched Region NOV20b 35..243 209/209 (100%) 3..211 209/209 ( 100%) Further analysis of the NOV20a protein yielded the following properties shown in Table 20C.
Table 20C. Protein Sequence Properties NOV20a PSort 0.8500 probability located in endoplasmic reticulum (membrane); 0.4400 probability analysis: located in plasma membrane; 0.3500 probability located in nucleus;
0.1000 probability located in mitochondria) inner membrane SignalP Cleavage site between residues 35 and 36 -analysis:
A search of the NOV20a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 20D.
Table 20D. Geneseq Results for NOV20a NOV20a Identities/

Geneseq Protein/Organism/Length Residues/Similarities for (Patent #, Expect Identifier Date] Match the Matched Value Residues Region AAB19126 Polypeptide isolated from14..506 382/503 (75%) 0.0 lymph node stromal cells of fsn -/- mice - 5..503 415/503 (81 fo) Mus sp, 503 aa. (W0200058463-Al, OS-OCT-2000]

AAU00670 Human TANGO 229 polypeptide238..506266/269 (98%) e-157 - Homo Sapiens, 715 aa. [W0200129088-A1, 447..715267/269 (98%) AAU00630 ' Novel human protein (NHP)1..243 241/243 (99%) e-138 sequence #3 - Homo Sapiens, 539 aa. [W0200129219-1..243 2421243 (99%) A1, 26-APR-2001) AAU00629 Novel human protein (NHP) 1..243 241/243 (99%) e-138 sequence #2 - Homo Sapiens, 586 aa. [W0200129219-48..290242/243 (99%) A 1, 26-APR-2001 ) AAU00628 Novel human protein (NHP) 53..243189/191 (98%) e-107 sequence #1 - Homo Sapiens, 487 aa. [W0200129219-1..191 190/191 (98%) A1, 26-APR-2001) In a BLAST search of public sequenceNOV20a datbases, the protein was found to have homology to the proteins shown in the BLASTP data in Table 20E.
Table 20E. Public BLASTP
Results for NOV20a Protein NOV20a Identities/

AccessionProtein/Organism/Length Residues/SimilaritiesExpect for Number Match the Matched Value ResiduesPortion Q9D4J3 4631413K11 RIK PROTEIN - 14..506 384/503 (76%)0.0 Mus musculus (Mouse), 503 aa. 5..503 416/503 (82%) Q9D696 4631413K11RIK PROTEIN - 53..506 353/464 (76%)0.0 Mus musculus (Mouse), 460 aa. 1..460 383/464 (82%) Q96NH2 CDNA FLJ30900 FIS, CLONE 352..506155/155 (100%)1e-89 FEBRA2005752 - Homo Sapiens1..155 155/155 (100%) (Human), 155 aa.

Q96PD2 ENDOTHELIAL AND SMOOTH 20..249 110/2361,46%)4e-51 MUSCLE CELL-DERIVED 51..286 148/236 (62%) - Homo Sapiens (Human), 775 aa.

Q91ZV3 ENDOTHELIAL AND SMOOTH 3..249 117/271 (43%)Se-50 MUSCLE CELL-DERIVED 13..283 154/271 (56%) NEUROPILIN-LIKE PROTEIN
- Mus musculus (Mouse), 769 aa.

PFam analysis predicts that the NOV20a protein contains the domains shown in the Table 20F.

Table 20F. Domain Analysis of NOV20a Identities/
Pfam Domain NOV20a Match Region Similarities Expect Value for the Matched Region CUB 41..147 34/117 (29%) 2.8e-20 73/117 (62%) Examule 21.
The NOV21 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 21A.
Table 21A. NOV21 Sequence Analysis SEQ ID NO: 73 X2570 by ~NOV21 a, ~CG97451-O1 DNA
Sequence GAAGTGGAAGTGGAGGTGGTGGGGGAGGTGGTCTCCTGGGGGGCCTGCTTGGTGGTGGi GGGTGGAGGGGGTGGCAGTGATCTGTTGGGTGGAGGCTTACTGGGTGGCAGTGGCAGC~
TACAGACGCATTGAATTCCCCCGAGGTGTTGGTGATATTCCCTACAATGACTTCCATGI
GAGATCCTTGAGTCCGAGGGAAGCATCAGGGACCTCCGAAACAGTGGCTATCGCAGTG
CCGAGAATGCATATGGAGGCCACAGGGGCCTCGGGCGATACAGGGCAGCACCTGTGGG
TCGCAGGCCAAGGTGGCCTGCTCGGCGGAGGTGGTCTCCTTGGTGATGGAGGACTTCT
TGGAGGAGGGGGTGTCCTGGGCGTGCTCGGCGAGGGTGGCATCCTCAGCACTGTGCAA~
GAACCGAGTCCTGGCCGACGTCCTCCCTGACTTGCTCTGCCCCATCGTGGATGTGGTGI
CCAAGTCCCAGCTGGCCATGTCTGCCAACTTCCTGGGCTCAGTGCTGACTCTACTGCA~
GAAGCAGCATGCTCTAGACCTGGATATCACCAATGGCATGTTTGAAGAGCTTCCTCCA
CCTGCCCACTTATCA
CAGAACCTCPAACGTGGGCAACTTTGATATTGGCCTCATGGAGGTGCTGGTGGAGAAG~

ATTTTTGACCTGGCATTCATGCCCGCAATGAACGCTGTGCTGGGTTCTGGCGTCCCTC

TCCCCAAAATCCTCAACATCGACTTTAGCAATGCAGACATTGACGTGTTGGAGGACCT

TTTGGTGCTGAGCGCATGAGTGACAGAGGCAGAGATGCTGCTGCAACTGGAAGAAGCT

GGAACCAGTCCCAGAGAGGCTCGGCCTGGAAACAGTCCCCTGCCCAGAGTCCCCTCAG

CCTCCATGACAGGTCCCTCCCTGGCCCCCCAACCCTCTTCCTCCCTTGCCCCAACCCT

GAGAAAGGGTCCAGCCACTACCCTGTTGGCAAACATTCCCTTCCATGGTCAGCCTGCC

AGGAGGAGGGGAGTCACCTTGGGGCTGGAGGCCTCTCAGACCCCATCCTGACAGCAGG

TTGAGTATTCCCACTTTCAATAAAAGACTCCACTTTCCCGGCACTTGTGACGAGTTTC

CATGAAGGACCCTCCTGA

ORF Start: ATG at ORF Stop: TGA at 2221 SEQ ID NO: 74 697 as MW at 71241.3kD

NOV2la, ~ MLQQSDALHSALREVPLGKARGDGGGPLLGGLLGGSGSGGGGGGGLLGGLLGGGGGGG

Protein SequenceSGGGLLGGGRHHYNDYRRIEFPRGVGDIPYNDFHVRGPPPVYTNGKKLDGIYQYGHIE

TNDNTAQLGGKYRYGEILESEGSIRDLRNSGYRSAENAYGGHRGLGRYRAAPVGRLHR

RELQPGEIPPGVATGAVGPGGLLGTGGMLAADGILAGQGGLLGGGGLLGDGGLLGGGG

VLGVLGEGGILSTVQGITGLRIVELTLPRVSVRLLPGVGVYLSLYTRVAINGKSLIGF

LDIAVEVNITAKVRLTMDRTGYPRLVIERCDTLLGGIKVKLLRGLLPNLVDNLVNRVL

ADVLPDLLCPIVDVVLGLVNDOLGLVDSLIPLGILGSVO__YTFSSLPLVTGEFLELDLN

TLVGEAGGGLIDYPLGWPAVSPKPMPELPPMGDNTKSQLAMSANFLGSVLTLLQKQHA

LDLDITNGMFEELPPLTTATLGALIPKVFQQYPESCPLIIRIQVLNPPSVMLQKDKAL

VKVLATAEVMVSQPKDLETTICLIDVDTEFLASFSTEGDKLMIDAKLEKTSLNLRTSN

VGNFDIGLMEVLVEKIFDLAFMPAMNAVLGSGVPLPKILNIDFSNADIDVLEDLLVLS

A

Further analysis of the NOV21 a protein yielded the following properties shown in Table 21B.
Table Z1B. Protein Sequence Properties NOVZla PSort 0.8500 probability located in endoplasmic reticulum (membrane); 0.4400 probability analysis: located in plasma membrane; 0.3033 probability located in microbody (peroxisome);
0.1000 probability located in mitochondria) inner membrane SignalP No Known Signal Sequence Predicted analysis:
A search of the NOV21 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 21 C.
Table 21C. Geneseq Results for NOV2la NOV2la Identities/

Geneseq Protein/Organism/Length [PatentResidues/Similarities Expect #, for Identifier Date] Match the Matched Value ResiduesRegion AAM05009 Peptide #3691 encoded by 1..188 ~ 188/188 e-110 probe for (100%) measuring breast gene expression 4..191 188/188 (100%) -Homo Sapiens, 191 aa. [WO200157270-A2, 09-AUG-2001 ]

AAM69485 Human bone marrow expressed1..188 I 88/188 ( e-1 probe 100%) I

encoded protein SEQ ID NO: 29791 4..191 188/188 (100%) -Homo Sapiens, 191 aa. [WO200157276-A2, 09-AUG-2001 ]

AAM57094 Human brain expressed single1..188 188/188 (100%)e-110 exon probe encoded protein SEQ ID NO: 4..191 188/188 (100%) 29199 - Homo sapiens, 191 aa.

[W0200157275-A2, 09-AUG-2001]

ABB21687 Protein #3686 encoded by 1..188 ~ 188/188 (100%)e-110 probe for measuring heart cell gene expression4..191 188/188 (100%) -Homo sapiens, 191 aa. [W0200157274-A2, 09-AUG-2001 AAG77922 Human new lipid binding 278..696165/420 (39%) 1 e-77 protein 3 -Homo Sapiens, 472 aa. [W0200179492- 62..469~
252/420 (59%) A2, 25-OCT-2001]

In a BLAST search of public sequence~dO
datbases, the V 21 a p.roWi~~
vYa~
found iv itav2 homology to the proteins shown in the BLASTP data in Table 21D.
Table 21D. Public BLASTP Results for NOV2la Protein NOV2la Identities/

AccessionProtein/Organism/Length Residues/Similarities Expect for Number Match the Matched Value ResiduesPortion CAD121S0 SEQUENCE 3 FROM PATENT 96..697 573/602 (95%)0.0 W00179269 - Homo sapiens 57..637 573/602 (95%) (Human), 637 aa.

CAD12149 SEQUENCE 1 FROM PATENT 136..697558/562 (99%)0.0 W00179269 - Homo Sapiens 53..614 559/562 (99%) (Human), 614 as (fragment).

CAClBoe DJ726C3.5 (ORTHOLOG OF ~ 229..697469/469 (100%)0.0 7 ~

POTENTIAL LIGAND BINDING 1..469 469/469 (100%) PROTEIN RY2G5 (RAT)) - Homo Sapiens (Human), 469 as (fragment).

Q05704 POTENTIAL LIGAND-BINDING 229..696426/469 (90%)0.0 PROTEIN - Rattus rattus 1..469 451/469 (95%) (Black rat), 470 as (fragment).

Q05701 POTENTIAL LIGAND-BINDING 243..696I 87/470 (39%)8e-83 PROTEIN - Rattus rattus 13..470 275/470 (57%) (Black rat), 473 aa.

PFam analysis predicts that the NOV21 a protein contains the domains shown in the Table 21 E.

Table 21E. Domain Analysis of NOV2la Identities/
Pfam Domain NOV2la Match Region Similarities Expect Value for the Matched Region Collagen 245..304 19/61 (31%) 0.72 24/61 (39%) LBP_BPI_CETP_C 512..697 51/197 (26%) 2.7e-11 111/197 (S6%) Example 22.
The NOV22 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 22A.
Table 22A. NOV22 Sequence Analysis SEQ ID NO: 7S 11 SO by NOV22a, GACTGCCTGGCAGGTGTGAAAGGCAGCGGTGGCCACAGAGGCGGTGGAGATGGCCTTC

CG97~S2-OI AGCGGTTCCCAGGCTCCCTATCTGAGCCCAGCCGTCCCCTTTTCTGGGACTATCCAAG

DNA

SequeriCe GGGGTCTCCAGGACGGATTTCAGATCACTGTCAATGGGGCCGTTCTCAGCTCCAGTGG

AACCAGGTTTGCTGTGGACTTTCAGACGGGCTTCAGTGGAAACGACATTGCCTTCCAC

TTCAACCCTCGGTTTGAAGACGGAGGGTATGTGGTGTGCAACACGAGGCAGAAAGGAA

GATGGGGGCCCGAGGAGAGGAAGATGCACATGCCCTTCCAGAAGGGGATGCCCTTTGA

CCTCTGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTAATGGTGAACGGGAGCCTCTTC

GTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCATCTCCGTCAATGGCT

CTGTGCAGCTGTCCTACATCAGCTTCCAGAATCCCCGCACAGTCCCCGTTCAGCCTGC

CTTCTCCACGGTGCCGTTCTCCCAGCCTGTCTGTTTCCCACCCAGGCCCAGGGGGCGC

AGACAAAAACCTCCCAGCGTGCGGCCTGCCAACCCAGCTCCCATTACCCAGACAGTCA

TCCACACGGTGCAGAGCGCCTCTGGACAGATGTTCTCTACTCCCGCCATCCCACCTAT

GATGTACCCCCACCCTGCCTATCCGATGCCTTTCATCACCACCATTCCGGGAGGGCTG

TACCCATCCAAGTCCATCATCCTGTCAGGCACTGTCCTGCCCAGTGCTCAGAGGTTCC

ACATCAACCTGTGCTCTGGGAGCCACATCGCCTTCCACATGAACCCCCGTTTTGATGA
_ ~

GAATGCTGTGGTCCGTAACACCCAGATCAACAACTCTTGGGGGTCTGAGGAGCGAAGT

CTGCCCCGAAAAATGCCCTTCGTCCGAGGCCAGAGCTTCTCGGTATGGATCTTGTGTG

AAGCTCACTGCCTCAAGGTGGCCGTGGATGGTCAGCACGTGTTTGAATACTACCATCG

CCTGAGGAACCTGCCCACCATCAACAAACTGGAAGTGGGTGGCGACATCCAGCTGACC

CACGTGCAGACATAGGCGGCTCCCTGGCCCTGGGGCCGGGGGCTGGGG

OItF Start: ATG at SO ORF Stop: TAG
at 111 S

SEQ 1D NO: 76 3SS as MW at 39S32.OIcD

NOV22a, MAFSGSQAPYLSPAVPFSGTIQGGLQDGFQITVNGAVLSSSGTRFAVDFQTGFSGNDI

CG978S2-OI A~~RFEDGGYWCNTRQKGRWGPEERKMHNIPFQKGMPFDLCFLVQSSDFKVMVNG

Protein SequenceSLFVQYFHRVPFHRVDTISVNGSVQLSYISFQNPRTVPVQPAFSTVPFSQPVCFPPRP

RGRRQKPPSVRPANPAPITQTVIHTVQSASGQMFSTPAIPPMMYPHPAYPMPFITTIP

GGLYPSKSIILSGTVLPSAQRFHINLCSGSHTAFHMNPRFDENAVVRNTQINNSWGSE

ERSLPRKMPFVRGQSFSVWILCEAHCLKVAVDGQHVFEYYHRLRNLPTINKLEVGGDI

QLTHVQT .

SEQ ID NO: 77 1460 by NOV22b, CTACAAAGGACTTCCTAGTGGGTGTGAAAGGCAGCGGTGGCCACAGAGGCGGCGGAGA

CG978S2-03 _GATGGCCTTCAGCAGTTCCCAGGCTCCCTACCTGAGTCCAGCTGTCCCCTTTTCTGGG

DNA

ACTATTCAAGGAGGTCTCCAGGACGGACTTCAGATCACTGTCAATGGGACCGTTCTCA

Sequence GCTCCAGTGGAACCAGGTTTGCTGTGAACTTTCAGACTGGCTTCAGTGGAAATGACAT
TGCCTTCCACTTCAACCCTCGGTTTGAAGACGGAGGGTATGTGGTGTGCAACACGAGG
CAGAAAGGAACATGGGGGCCCGAGGAGAGGAAGACACACATGCCTTTCCAGAAGGGGA
TGCCCTTTGACCTCTGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTGATGGTGAACGG
GATCCTCTTCGTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCATCTCC
GTCAATGGCTCTGTGCAGCTGTCCTACATCAGCTTCCAGCCTCCCGGCGTGTGGCCTG
CCAACCCGGCTCCCATTACCCAGACAGTCATCCACACAGTGCAGAGCGCCCCTGGACA
GATGTTCTCTACTCCCGCCATCCCACCTATGGTG'PACCCCCACCCCGCCTATCCGATG
ACCAGCTGTCTGCTCCTGGTGGGAGGTGGCCCTCCTCAGCCCCTCCTCTCTGACCTTT
AACCTCACTCTCACCTTGCACCGTGCACCAACCCTTCACCCCTCCTGGAAAGCAGGCC
TCCTTTCCCAGTGTCCTTAAAATAAAGAAATGAAAATGCTTGTTGG
OItF Start: ATG at 60 ORF Stop: TGA at 798 SEQ ID NO: 78 246 as MW at 26802:6kD
NOV22b, MAFSSSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFSGNDI
CG97852-O3 ~FNPRFEDGGYWCNTRQKGTWGPEERKTHMPFQKGMPFDLCFLVQSSDFKVMVNG
Pi'Oteln Sequence ILFVQYFHRVPFHRVDTISVNGSVQLSYISFQPPGVWPANPAPITQTVIHTVQSAPGQ
MFSTPAIPPMVYPHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRCGSCVKLTASR
WPWMVSTCLNTTIA
Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 22B.
Table 22B. Comparison of NOV22a against NOV22b.
Protein ~eqr,~ence I NOV22a Residues/ Identities/ E
Match Residues Similarities for the Matched Region NOV22b 1..253 208/253 (82%) 1..221 211/253 (83%) Further analysis of the NOV22a protein yielded the following properties shown in Table 22C.
Table 22C. Protein Sequence Properties NOV22a PSort 0.6400 probability located in microbody (peroxisome); 0.3267 probability located in analysis: Iysosome (lumen); 0.3000 probability located in nucleus; 0.1000 probability located in mitochondria) matrix space SignaIP No Known Signal Sequence Predicted analysis:

A search of the NOV22a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 22D.
Table 22D. Geneseq Results for NOV22a NOV22a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAE13847 Human lung tumour-specific1..355 ~ 338/355 0.0 protein (95%) 21871 - Homo Sapiens, 378 24..378 345/355 (96%) aa.

[W0200172295-A2, 04-OCT-2001]

AAE13847 Human lung tumour-specific1..355 338/355 (95%)0.0 protein 21871 - Homo sapiens, 378 24..378 ~
aa. 345/355 (96%) [W0200172295-A2, 04-OCT-2001]

AAY06997 Galectin-9 protein sequence1..355 338/355 (95%)0.0 - Homo Sapiens, 355 aa. [W09904265-A2,1..355 345/355 (96%) JAN-1999] ~

AAW85664 Galectin-9 like protein 1..355 ~ 338/355 0.0 - Homo sapiens, (95%) 355 aa. [W099I0490-A1, 1..355 ~ 345/355 04-MAR- (96%) 1999]

AAY56802 Human eosinophil chemotactic1..355 [ 305/355 e-179 factor (85%) (ecalectin)~- Homo Sapiens,1..323 ~ 312/355 323 aa. (86%) [W09962556-Al, 09-DEC-1999]

In a BLASTsearch of public sequence datbases, the NOV22a protein was found to have homology to the proteins shown in the BLASTP data in Table 22E.
_~.. . Fable 22E. Public BLASTP Results for NO~%2~a NOV22a Protein Identities/

Accession Protein/Organism/Length Residues/Similarities Expect for the Number Matched PortionValue Residues Q9NQ58 GALECTIN-9 - Homo Sapiens 1..355 344/355 (96%) 0.0 (Human), 355 aa. 1..355 348/355 (97%) 000182 Galectin-9 (HOM-HD-21) .1..355 338/355 (95%) 0.0 (Ecalectin) - Homo sapiens (Human),1..355 345/355 (96%) 355 aa.

Q9XSM9 URATE TRANSPORTER/CHANNEL PROTEIN, 1..345263/345 e-(76%) ISOFORM~(UATP,I) - Sus scrofa (Pig), 349 1..345292/345 160 aa. (84%) P97840 Galectin-9 (36 kDa beta-galactoside 1..355251/355 e-binding lectin) (70%) (Urate transporter/channel) (UAT) - Rattus 1..354286/355 151 norvegicus (79%) (Rat), 354 aa.

008573 Galectin-9 - Mus musculus (Mouse), 1..355244/355 a 353 aa. 68%) ( 1..353~ 285/355 146 (79%) PFam analysis predicts that the NOV22a protein contains the domains shown in the Table 22F.
Table 22F. Domain Analysis of NOV22a Identities/ T .
Pfam Domain NOV22a Match Region Similarities ' .~uR~r2Ci i'aiu2 for the Matched Region Gal-bind_lectin 16..147 49/139 (35%) 1.2e-43 106/139 (76%) Gal-bind_lectin 226..355 ~ 5I/142 (36%) 7.3e-39 102/142 (72%) Example 23.
The NOV23 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 23A.

ORF Start: at 2 ORF Stop: TGA at _989 _ SEQ ID NO: 80 329 as MW at 37I30.91cD
NOV23a, QFLLLALLLPGGDNADASQEHVSFFiAIQIFSFVNQSWARGQGSGWLDELQTHGWDSES

Protein SequenceE~SGKSPEGFFQVAFNGLDLLSFQNTTWVPSPGCGSLAQSVCHLLNHQYEGVTETVY

VTWNn2NEQEQLGTKHGDILPNADGTWYLQVILEVASEEPAGLSCRVRHSSLGGQDIIL

YWGHFiFSMNWIALVVIVPLVILIVLVLWFKKHCSYQDIL

SEQ ID NO: 81 1116 by NOV23b, ACAGAGATCAGCAAACAGCTTTTCTGAGAGAAAGAAACATCTGCAAATGACATGCTGT

DNA

Sequence CCAGGAACACGTCTCCTTCCATGTCATCCAGATCTTCTCATTTGTCAACCAATCCTGG

GCACGAGGTCAGGGCTCAGGATGGCTGGACGAGTTGCAGACTCATGGCTGGGACAGTG

CAAGACCATGCAAGTCAAGATTACTCGAAATATCCCTTTGAAGTACAGGTGAAAGCGG
GCTGTGAGCTGCATTCTGGAAAGAGCCCAGAAGGCTTCTTTCAGGTAGCTTTCAACGG
ATTAGATTTACTGAGTTTCCAGAATACAACATGGGTGCCATCTCCAGGCTGTGGAAGT
TTTGGGTGACATGGA
TCTTCCTAATGCTGA
TCCTCTACTGGGCTCATATCAGGACATCCTGTGAGACTCTTCCCCCTGACTCCCCCAT
TGTGTTAAGAACCCAGCAACCCAGGAGCCTAGTACAATATAGTGATGCCATCCCGTCG
ACTCTCCATTTAAATTGTTTCTCTTTCTGCATAATAAACATTTGTTAATAAAAACCAA
ORF Start: ATG at S2 ORF Stop: TAA at 1090 SEQ 1D NO: 82 346 as MW at 38907.71eD
NOV23b, MLFLQFLLLALLLPGGDNADASQEHVSFHVIQIFSFVNQSWARGQGSGWLDELQTHGW

Protein Sequence ~GCELHSGKSPEGFFQVAFNGLDLLSFQNTTWVPSPGCGSLAQSVCHLLNHQYEGVT
ETVYNLIRSTCPRFLLGLLDAGKMYVHRQVRPEAWLSSRPSLGSGQLLLVCHASGFYP
KPVWVTWNIRNEQEQLGTKHGDILPNADGTWYLQVILEVASEEPAGLSCRVRHSSLGGQ
DIILX~1AHTRTSCETLPPDSPIVLRTQQPRSLVOYSDAIPSTLHLNCFSFCIINIC
Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 23B.
Table 23B.
Comparison of NOV23a against NOV23b.

Protein SequenceNOV23a Residues/Identities/

Match ResiduesSimilarities for the Matched Region NOV23b 10..294 282/285 (98%) 14..298 282/285 (98%) Further analysis of the NOV23a protein yielded the following properties shown in Table 23C.

Table 23C. Protein Sequence Properties NOV23a PSort 0.4600 probability located in plasma membrane; 0.3053 probability located in analysis: microbody (peroxisome); 0.3000 probability located in lysosome (membrane); 0.2800 probability located in endoplasmic reticulum (membrane}
SignalP Cleavage site between residues 16 and 17 analysis:
A search of the NOV23a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 23D.
Table 23D. Geneseq Results for NOV23a 1 NOV23aId~endties/ 1 Geneseq ProteinlOrganism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion ABG13799Novel human diagnostic protein28..194 162/167 (97%)3e-95 #13790 - Homo Sapiens, 681 aa. 515..681165/167 (98%) [WO200175067-A2, 11-OCT-2001]

ABG13799Novel human diagnostic protein28..194 162/167 (97%)3e-95 #13790 - Homo Sapiens, 681 aa. 515..681165/167 (98%) [W0200175067-A2, 11-OCT-2001]

AAG00593Human secreted protein, I..60 56/60 (93%} 6e-27 SEQ ID NO:

4674 - Homo sapiens, 64 5..64 57/60 (94%) aa.

[EP1033401-A2, 06-SEP-2000]

AAY94507Chicken BFIV 12 class I~MHC110..31758/209 (27%)2e-16 protein -Gallus gallus, 355 aa. [IJS6075125-A,114..315102/209 (48%) 13-JIJN-2000]

AAP83149Probe F10-encoded protein 110..31758/209 (27%)2e-16 of 1VIHC

class I of chicken - Gallus115..316102/209 (48%) gallus, 345 aa.

[W08809386-A, O1-DEC-1988]

In a BLAST search of public sequence datbases, the NOV23a protein was found to have homology to the proteins shown in the BLASTP data in Table 23E.

Table 23E. Public BLASTP
Results for NOV23a Protein NOV23a Identities/

Accession Protein/Organism/Length Residues/ SimilaritiesExpect for Number Match the Matched Value Residues Portion P29017 T-cell surface glycoprotein1..329 326/329 (99%)0.0 CDlc ~

precursor (CD I c antigen)5..333 326/329 (99%) - Homo Sapiens (Human), 333 aa.

Q9QZY6 T-cell surface glycoprotein1..328 216/328 (65%)e-126 CDIc3 precursor (CD1-c3 antigen)5..331 253/328 (76%) - Cavia porcellus (Guinea pig), 332 aa.

Q9QZY8 T-cell surface glycoprotein2..328 212/327 (64%)~ e-121 CDlcl precursor (CD 1-c 1 antigen)6..3? I 245!327 (74".) - Cavia porcellus (Guinea pig), 332 aa.

Q9QZY7 T-cell surface glycoprotein3 1..328 197/328 (60%)e-112 CDlc2 precursor (CDI-c2 antigen)5..331 239/328 (72%) - Cavia porcellus (Guinea pig), 332 aa.

P29016 T-cell surface glycoprotein2..328 197!328 (60%)e-110 CDlb precursor (CD1 b antigen)6..332 240/328 (73%) - Homo sapiens (Human), 333 aa.

PFam analysis predicts that the NOV23a protein contains the domain shown in the Table 23F.
Table 23F. Domain Analysis of NOV23a Identities/ . I 1 Pfam Domain NOV23a Match Region Similarities Expect Value for the Matched Region ig 214..278 15/67 (22%) ~ 4.4e-07 48167 (72%) Eaamule 24.
The NOV24 clone was analyzed, and the nucleotide=and encoded poIypeptide sequences are shown in Table 24A.
Table 24A. NOV24 Sequence Analysis SEQ ID NO: 83 ~ 1497 by CG99608-O1 A_CATGGAGGGGAGCGCGAGCCCCCCGGAAAAGCCCCGCGCCCGCCCTGCGGCTGCCGT
DNA

Sequence GCTGTGCCGGGGCCCGGTAGAGCCGCTGGTCTTCCTGGCCAACTTTGCCTTGGTCCTG

CAGGGCCCGCTCACCACGCAGTATCTGTGGCACCGCTTCAGCGCCGACCTCGGCTACA

ATGGCACCCGCCAAAGGGGGGGCTGCAGCAACCGCAGCGCGGACCCCACCATGCAGGA

AGTGGAGACCCTTACCTCCCACTGGACCCTCTACATGAACGTGGGCGGCTTCCTGGTG

GGGCTCTTCTCGTCCACCCTGCTGGGAGCTTGGAGCGACAGTGTGGGCCGCCGCCCGC

TGCTAGTGCTGGCCTCGCTGGGCCTGCTGCTCCAGGCCCTAGTGTCCGTTTTTGTGGT

GCAGCTGCAGCTCCACGTCGGCTACTTCGTGCTGGGTCGCATCCTTTGTGCCCTCCTC

GGCGACTTCGGTGGCCTTCTGGCTGCTAGCTTTGCGTCCGTGGCAGATGTCAGCTCCA

GTCGCAGCCGCACCTTCCGGATGGCCCTGCTGGAAGCCAGCATCGGGGTGGCTGGGAT

GCTGGCAAGCCTCCTCGGGGGCCACTGGCTCCGGGCCCAGGGTTATGCCAACCCCTTC

TGGCTGGCCTTGGCCTTGCTGATAGCCATGACTCTCTATGCAGCTTTCTGCTTTGGTG

AGACCTTAAAGGAGCCAAAGTCCACCCGGCTCTTCACGTTCCGTCACCACCGATCCAT

TGTCCAGCTCTATGTGGCTCCCGCCCCAGAGAAGTCCAGGAAACATTTAGCCCTCTAC

TCACTGGCCATCTTCGTGGTGATCACTGTGCACTTTGGGGCCCAGGACATCTTAACCC

TTTATGAACTAAGCACACCCCTCTGCTGGGACTCCAAACTAATCGGCTATGGTTCTGC

AGCTCAGCATCTCCCCTACCTCACCAGCCTGCTGGCCCTGAAGCTCCTGCAGTACTGC

CTGGCCGATGCCTGGGTAGCTGAGATCGGCCTGGCCTTCAACATCCTGGGGATGGTGG

TCTTTGCCTTTGCCACTATCACGCCTCTCATGTTCACAGGATATGGGTTGCTTTTCCT

GTCATTAGTCATCACACCTGTCATCCGGGCTAAACTCTCCAAGCTGGTGAGAGAGACA

GAGCAGGGTGCTCTCTTTTCTGCTGTGGCCTGTGTGAATAGCCTGGCCATGCTGACGG

CCTCCGGCATCTTCAACTCACTCTACCCAGCCACTCTGAACTTTATGAAGGGGTTCCC

CTTCCTCCTGGGAGCTGGCCTCCTGCTCATCCCGGCTGTTCTGATTGGGATGCTGGAA

AAGGCTGATCCTCACCTCGAGTTCCAGCAGTTTCCCCAGAGCCCCTGATCTGCCTGGA

CCAGAAGACAGAGGGCAAGAGGAGCAAAGTGAACACCAAGCAACTGG

ORF Start: ATG at 61 ORF Stop:
TGA at 143 8 SEQ ID NO: 84 459 as MW at 49769.9kD

NOV24a, MEGSASPPEKPRARPAAAVLCRGPVEPLVFLANFALVLQGPLTTQYLWHRFSADLGYN

Protein SequenceL~SLGLLLQALVSVFWQLQLHVGYFVLGRILCALLGDFGGLLAASFASVADVSSS

RSRTFRMALLEASIGVAGMLASLLGGHWLRAQGYANPFWLALALLIAMTLYAAFCFGE

TLKEPKSTRLFTFRHHRSIVQLYVAPAPEKSRKHLALYSLAIFWITVHFGAQDILTL

YELSTPLCWDSKLIGYGSAAQHLPYLTSLLALKLLQYCLADAWVAEIGLAFNILGMW

FAFATITPLMFTGYGLLFLSLVITPVIRAKLSKLVRETEQGALFSAVACVNSLAMLTA

SGIFNSLYPATLNFMKGFPFLLGAGLLLIPAVLIGMLEKADPALEFQQFPQSP

Further analysis of the NOV24a protein yielded the following properties shown in Table 24B.
Table 24B. Protein Sequence Properties NOV24a PSort 0.8000 probability located in plasma membrane; 0.4000 probability located in Golgi analysis: body; 0.3000 probability located in endoplasmic reticulum (membrane); 0.3000 probability located in microbody (peroxisome) SignalP No Known Signal Sequence Predicted analysis:
A search of the NOV24a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 24C.

Table 240. Geneseq Results for NOV24a 1 NOV24a Identities/

Geneseq Protein/OrganismJLength Residues/SimilaritiesExpect [Patent #, for Identifier~ Date] Match the Matched Value Residues Region AAE04906Human transporter and ion 36..438 139/420 (33%)9e-54 channel-19 (TRICH-19) protein - Homo 1..415 212/420 (50%) Sapiens, 445 aa. [W020014b258-A2, 200I ]

AAB41967~ Human ORFx ORF1731 polypeptide181..281 101/101 (100%)Se-52 sequence SEQ ID N0:3462 1..101 101/101 (100%) - Homo sapiens, 101 aa. [W0200058473-A2, OS-OCT-2000]

AAU14370Human novel protein #241 ~ 119..45986/365 (23%)Se-21 - Homo ~ ~

~ 1..358 156/365 (42%) -Sapiens, 365 aa. [WO200155437-A2, AAU 14134Human novel protein #5 - 119..459 86/365 (23%)Se-21 Homo Sapiens, 365 aa. [W0200155437-A2, 1..358 156/365 (42%) 2001]

ABB59118~ Drosophila melanogaster 25..443 101/440 (22%)3e-20 polypeptide SEQ ID NO 4146 - Drosophila403..838 1941440 (43%) melanogaster, 856 aa. [W0200171042-A2, 27-SEP-2001 .... ... .... ... .....
.. . _. . ......

In a BLAST search of public sequence datbases, the NOV24a protein was found to have homology to the proteins shown in the BLASTP data in Table 24D.
Table 24D. Public BLASTP
Results for NOV24a Protein NOV24a Ide~atitiQS/

AccessionProtein/Organism/Length Residues/SimilaritiesExpect for Number Match the Matched Value ResiduesPortion Q96NT5 CDNA FLJ30107 FIS, CLONE 1..459 459/459 (100%)0.0 BNGH41000198, WEAKLY SIMILAR1..459 459/459 (100%) TO TETRACYCLINE RESISTANCE

PROTEIN, CLASS E - Homo sapiens (Human), 459 aa.

Q96FL0 SIMILAR TO RIKEN CDNA 1..459 431/459 (93%)0.0 I 110002008 GENE - Homo SapiensI ..431 431/459 (93%) (Human), 431 aa.

Q9D1P1 1110002C08RIK PROTEIN-Mus 1 1..459399/459 (86%)0.0 musculus (Mouse), 459 aa. ~ 1..459418/459 (90%) Q28720 HYPOTHETICAL 31.9 KDA PROTEIN~ 181..459242/279 (86%)e-138 - Oryctolagus cuniculus (Rabbit),1..279 260/279 (92%) 293 aa.

WO 02/090504 PCT/US02/14342 ..
. . . ....._....._..__._. ..........._..
__._.~._._......................_......_. . . .. . .
.~,y".~,..._........_............ _.._",~,~",..........._.._.....
AAH24522 SIMILAR TO RIKEN CDNA t 264..459 ; 178/196 (90%) 3e-98 1110002008 GENE - Mus musculus ~ 1..196 186/196 (94%) (Mouse), 196 as (fragment).
PFam analysis predicts that the NOV24a protein contains the domains shown in the Table 24E.
Table 24E.
Domain Analysis of NOV24a Identities/

Pfam DomainNOV24a Match Similarities Eacpect Region Value for the Matched Region Sugar_tr 21..459 73/S19 (14%) 0.017 26$/S 19 (S 1 %) Eacample 25.
The NOV2S clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 25A.
Table 25A. NOV25 Sequence Analysis SEQ ID NO: 85 564 by NOV2Sa, TCCCTCATGTATGGCAAGAGCTCTACTCGTGCGGTGCTTCTTCTCCTTGGCATACAGC

CG99674-O1 T~~GCTCTTTGGCCTATAGCAGCTGTGGAAATTTATACCTCCCGGGTGCTGGAGGC
DNA

Sequence TGTTAATGGGACAGATGCTCGGTTTAAGGACCGGGTGTCTTGGGATGGGAATCCTGAG

CGGTACGATGCCTCCATCCTTCTCTGGAAACTGCAGTTCGACGACAATGGGACATACA

CCTGCCAGGTGAAGAACCCACCTGATGTTGATGGGGTGATAGGGGAGATCCGGCTCAG

CGTCGTGCACACTGTACGCTTCTCTGAGATCCACTTCCTGGCTCTGGCCATTGGCTCT

GCCTGTGCACTGATGATCATAATAGTAATTGTAGTGGTCCTCTTCCAGCATTACCGGA

AAAAGCGATGGGCCGAAAGAGCTCATAAAGTGGTGGAGATAAAATCAAAAGAAGAGGA

AAGGCTCAACCAAGAGAAAAAGGTCTCTGTTTATTTAGAAGACACAGACTAACAATTT

TAGATGGAAGCTGAGATGATTTCCAAGAACAAGAACCCTAGT

ORF Start: ATG at ORF Stop:
7 TAA at SEQ ID NO: 86 169 as MW at 19261.11cD

NOV25a, MYGKSSTRAVLLLLGIQLTALWPIAAVEIYTSRVLEAVNGTDARFKDRVSWDGNPERY

Protein Sequence~MIIIVIVWLFQHYRKKRWAERAHKVVEIKSKEEERLNQEKKVSVYLEDTD

Further analysis of the NOV2Sa protein yielded the following properties shown in Table 25B.
Table 25B. Protein Sequence Properties NOV25a PSort 0.4600 probability located in plasma membrane; O.I000 probability located in analysis: endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in outside SignalP ~ Cleavage site between residues 27 and 28 analysis:
A search of the NOV25a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 25C.
Table 25C. Geneseq Results for NOV25a NOV25a Identities/

Geneseq Protein/Organism/Length [PatentResidues/SimilaritiesExpect #, for IdentifierDate] Match the Matched Value ResiduesRegion AAB65275Human PRO! 192 (UNQ606) protein1..169 169/215 (78%)9e-87 sequence SEQ ID N0:389 - 1..215 169/215 (78%) Homo Sapiens, 215 aa. [W0200073454-A1, DEC-2000]

AAU12415Human PRO1192 polypeptide 1..169 169/215 (78%)9e-87 sequence -Homo Sapiens, 215 aa. [W0200140466-1 ..21 169/215 (78%) S

A2, 07-JUN-2001 ]

AAY66752Membrane-bound protein PROI I ..169 169/215 (78%)9e-87 Homo Sapiens, 215 aa. [W09963088-A2,I ..215 169/215 (78%) 09-DEC-1999]

AAB33448Human PRO1192 protein UNQ606~ 1..169169/215 (78%)9e-87 SEQ

ID NO:163 - Homo sapiens, 1..21 169/215 (78%) 215 aa. S

[W0200053758-A2, 14-SEP-2000]

AAY41673Human channel-related molecule1..169 169/215 (78%)9e-87 HCRM-1 - Homo Sapiens, 215 aa. 1..215 169/215 (78%) [W09943807-A2, 02-SEP-1999]

In a BLAST search of public sequence datbases, the NOV25a protein was found to have homology to the proteins shown iri the BLASTP data in Table 25D.
Table 25D. Public BLASTP
Results for NOV25a Protein NOV25a Identities/

AccessionProtein/Organism/Length Residues/Similarities Expect for Number Match the Matched Value ResiduesPortion 060487 Epithelial V-like antigen I ..169 I 69/215 (78%)2e-86 1 precursor -Homo sapiens (Human), 215 I ..215 169/215 (78%) aa.

070255 Epithelial V-like antigen I ..169 134/215 (62%)Se-66 I precursor -Mus musculus (Mouse), 21 I ..215 146/215 (67%) S aa.

Q9I WI4 EPITHELIAL V-LIKE ANTIGEN I ..I 133/215 (6I 3e-65 - Mus 69 %) musculus (Mouse), 215 aa. I ..215 1451215 (66%) , P37301 Myelin PO protein precursor (Myelin38/94 (40%) 2e-12 45..138 protein zero) (Myelin peripheral 53/94 (SS%) protein) 95..188 (MPP) - Gallus gallus~(Chicken), 249 aa.

91406 IPI - Salmo s , 202 aa. 45..136 36/92 (39%) 3e-12 Q ~ p ~

88..179 Sl/92 (SS%) PFam analysis predicts that the NOV2Sa protein contains the domain shown in the Table 25E.
Table 25E. Domain Analysis of NOV25a Identities/
Pfam Domain NOV25a Match Region Similarities Expect Value for the Matched Region ig 39..79 ~ 10/42 (24%) 0.0023 34%42 (81%) Example 26.
S The NOV26 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 26A.
Table 26A.
NOV26 Sequence Analysis SEQ ID NO: 87 820 by NOV26a, A_TGGGCCACTTCCTCTCCAGTCCCTCCTGCAGCGTCTCTGCTCTGGGCCCTGCCATCT

DNA

Sequence ~TTCCAAATTTCAGAGGGACCTGGTGACCCTGAGAACAGATTTTAGCAACTTCA

CCTCAAACACTGTGGCGGAGATCCAGGCACTGACTTCCCAGGGCAGCAGCTTGGAAGA

AACGATAGCATCTCTGAAAGCTGAGGTGGAGGGTTTCAAGCAGGAACGGCAGGCAGTT

CATTCTGAAATGCTCCTGCGAGTCCAGCAGCTGGTGCAAGACCTGAAGAAACTGACCT

GCCAGGTGGCTACTi TCAACAACAATGCCTCCAC'I'GAAGGGACCTvCTGCCt:iGTCAA

CTGGGTGGAGCACCAAGACAGCTGCTACTGGTTCTCTCACTCTGGGATGTCCTGGGCC

GAGGCTGAGAAGTACTGCCAGCTGAAGAACGCCCACCTGGTGGTCATCAACTCCAGGG

AGGAGCAGAATTTTGTCCAGAAATATCTAGGCTCCGCATACACCTGGATGGGCCTCAG

TGACCCTGAAGGAGCCTGGAAGTGGGTGGATGGAACAGACTATGCGACCGGCTTCCAG

AACTGGAAGCCAGACCAGCCAGACGACTGGCAGGGGCACGGGCTGGGTGGAGGCGAGG

ACTGTGCTCACTTCCATCCAGTCGGCAGGTGGAATGACGACGTCTGCCAGAGGCCCTA

CCACTGGGTCTGCGAGGCTGGCTTGGGTCAGACCAGCCAGGAGAGTCACTGAGGTACC

TTTGGTGG

ORF Start: at 3 ORF Stop:
TGA
at 804 SEQ ID NO: 88 267 as MW at 29924.3kD

NOV26a, GPLPLQSLLQRLCSGPCHLLLSLGLGFLLLVIICWGFQNSKFQRDLWLRTDFSNFT

CG99732-02 SN'~~IQALTSQGSSLEETIASLKAEVEGFKQERQAVHSEMLLRVQQLVQDLKKLTC

Protein SequenceQ~'T~'~TEGTCCPVNWVEHQDSCYWFSHSGMSWAEAEKYCQL'r~IAHLWINSRE

EQNFVQKYLGSAYTWMGLSDPEGAWKWVDGTDYATGFQNWKPDQPDDWQGHGLGGGED

CAHFHPVGRWNDDVCQRPYHWVCEAGLGQTSQESH

SEQ ID NO: 89 1072 by NOV26b, CTCCATTTCAGCTGTGACAACCTCAGAGCCGTGTTGGCCTAAGCATGACAAGGACGTA

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 26B.
Table 26B. Comparison of NOV26a against NOV26b.
Protein Sequence NOV26a Residues! Identities/
Match Residues Similarities for the Matched Region 'NOV26b ~ l:.lfi'1 253%297 (85%) 23..319 253/297 (85%) Further analysis of the NOV26a protein yielded the following properties shown in Table 26C.
Table 26C. Protein Sequence Properties NOV26a Psort 0.7900 probability located in plasma membrane; 0.6756 probability located in analysis: microbody (peroxisome); 0.3000 probability located in Golgi body;
0.2000 probability located in endoplasmic reticulum (membrane) SignaIP Cleavage site between residues 38 and 39 analysis:

A search of the NOV26a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 26D.
Table 26D. Geneseq Results for NOV26a NOV26a Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAW88125 Primate DCMP2 C-lectin family1..267 263/294 (89%)e-155 gene protein sequence - Mammalia,23..316 263/294 (89%) 316 aa.

[W09902562-A1, 21-JAN-1999]

AAW88129 Variant primate DCMP2 C-lectin1..267 246/270 (91%)e-145 family gene protein sequence - 23..273 246/270 (91 Mammalia, 273 %) aa. [W09902562-AI, 21-JAN-1999]

AAW 15245Asialoglycoprotein receptor1..265 161/265 (60%)1 e-98 HI - Homo Sapiens, 291 aa. [EP773289-A2,24..287 202/265 (75%) MAY-1997]

AAW15250 Asialoglycoprotein receptor1..265 148/265 (55f)5e-88 HI

cytoplasmic+extracellular 24..270 188/265 (70%) domains -Chimeric Homo Sapiens, 274 aa.

[EP773289-A2, 14-MAY-1997]

AAW15249 Asialoglycoprotein receptor37..265 135/229 (58%)1e-83 Hl extracellular domain - ChimericI ..228 173/229 (74%) Homo Sapiens, 232 aa. [EP773289-A2, MAY-1997]

In a BLAST search of public sequence datbases, the NOV26a protein was found to have homology to the proteins shown in the BLASTP data in Table 26E.
Table 26E. Public BLASTP
Results for NOV26a .

Protein NOV26a Identities/

AccessionProtein/Organism/Length Residues/Similarities Expect for Number Match the Matched Value ResiduesPortion Q14538 MACROPHAGE LECT1N 2 - Homo1..267 263/270 (97%)e-158 .

Sapiens (Human), 292 aa. 23..292 263/270 (97%) BAB83508 ASIALOGLYCOPROTE1N 1..265 161/265 (60%)3e-98 RECEPTOR 1 - Homo Sapiens 24..287 202/265 (75%) (Human), 291 aa.

P07306Asialoglycoprotein receptor 1 (Hepatic1..265161/265 3e-lectin H1) 23..286(60%) 98 (ASGPR) (ASGP-R) - Homo sapiens (Human), 202!265 290 aa. (75%) Q91Y84ASIALOGLYCOPROTEIN RECEPTOR MAJOR 1..263149/263 le-(56%) SUBUNIT (ASIALOGLYCOPROTEIN RECEPTOR 23..284197/263 91 1 ) - (74%) Mus musculus (Mouse), 284 aa.

LNRTL Hepatic lectin - rat, 284 aa. 1..263150/263 3e-(57%) 23..284194/263 91 (73%) PFam analysis predicts that the NOV26a protein contains the domains shown in the Table 26F.
Table 26F. Domain Analysis of NOV26a Identities/
Pfam Domain NOV26a Match Region Similarities Expect v slue for the Matched Region lectin_c 149..257 41/127 (32%) 3e-45 94/127 (74%) Example 27.
The NOV27 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 27A.

Further analysis of the NOV27a protein yielded the following properties shown in Table 27B.
Table 27B. Protein Sequence Properties NOV27a PSort 0.4600 probability located in plasma membrane; 0.1000 probability located in analysis: endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in outside SignalP Cleavage site between residues 27 and 28 analysis:
A search of the NOV27a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 27C.
Table 27C. Geneseq Results for NOV27a NOV27a Identities/

Geneseq Protein/Organism/Length [PatentResidues/SimilaritiesExpect #, for IdentifierDate) Match the Matched Value ResiduesRegion ABG037941 Novel human diagnostic 52..178 69/134 (S1%)4e-21 protein #3785 -Homo Sapiens, 150 aa. [W0200175067-25..150 74/134 (54%) A2, I1-OCT-2001]

ABG03794Novel human diagnostic protein52..178 69/134 (S1%)4e-21 #3785 -Homo Sapiens, 150 aa. [W0200175067-25..150 74/134 (54%) A2, 11-OCT-2001]

AAB88581Human hydrophobic domain 44..197 52/155 (33%)1e-13 containing protein clone HP1072? #65 39..170 74/15 (47,il -.Homo Sapiens, 183 aa. [W0200112660-A2, AAY13464Human diaphanous polypeptide83..203 32/121 (26%)0.004 (Dial) -Homo Sapiens, 1248 aa. [W09922028-A1,605..71541/121 (33%) 06-MAY-1999]

ABG21919Novel human diagnostic protein79..203 ~ 38/125 0.006 #21910 - (30%) Homo sapiens, 325 aa. [W0200175067-74..191 ' 44/125 (34%) A2, I1-OCT-200I]

In a BLAST search of public sequence datbases, the NOV27a protein was found to have homology to the proteins shown in the BLASTP data in Table 27D.

Table 27D. Public BLASTP
Results for NOV27a Protein NOV27a Identities/

AccessionProtein/Organism/Length Residues/Similarities Expect for Number Match the Matched Value ResiduesPortion Q9D702 2310043I08RIK PROTEIN 1..196 141/196 (71%)6e-76 - Mus musculus (Mouse), 196 1..196 151/196 (76%) aa.

Q8UW65 P8F7 - Xenopus laevis 38..16$ 67/132 (50%) 3e-31 (African clawed frog), 229 as (fragment).59..183 87/132 (65%) CAC33296 SEQUENCE 85 FROM PATENT 44..197 52/155 (33%) 2e-13 W00112660 - Homo sapiens 39..170 74/I55 (47%) (Human), 183 aa.

Q96NA7 CDNA FLJ31166 FIS, CLONE 44..197 ~ 52/155 (33%)2e-13 KIDNE1000143 - Homo sapiens19..150 74/155 (47%) (Human), 163 aa.

Q9ASK4 HYPOTHETICAL 72.7 KDA 85..203 39/135 (28%) 0.007 PROTEIN - Oryza sativa 99..228 50/135 (36%) (Rice), 698 aa.

PFam analysis predicts that the NOV27a protein contains the domains shown in the Table 27E.
Table 27E. Domain Analysis of NOV27a Identities/
Pfam Domain NOV27a Match Region Similarities Expect Value for the Matched Region Example B: Sequencing Methodology and Identification of NOVX Clones 1. GeneCallingTM Technology: This is a proprietary method of performing differential gene expression profiling between two or more samples developed at GuraGen and described by Shimkets, et al., "Gene expression analysis by transcript profiling coupled to a gene database query" Nature Biotechnology 17:198-803 (1999). cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then digested with up to as many as I20 pairs of restriction enzymes and pairs of linker-adaptors specific for each pair of restriction enzymes were ligated to the appropriate end. The restriction digestion generates a mixture of unique cDNA gene fragments.
Limited PCR amplification is performed with primers homologous to the linker adapter sequence where one primer is biotinylated and the other is fluorescently labeled. The doubly labeled material is isolated and the fluorescently labeled single strand is resolved by capillary gel electrophoresis. A computer algorithm compares the electropherograms from an experimental and control group for each of the restriction digestions. This and additional sequence-derived information is used to predict the identity of each differentially expressed gene fragment using a variety of genetic databases. i ne identity of ts~:e gene fray en t is confizmed by additional, gene-specific competitive PCR or by isolation and sequencing of the gene fragment.
IS
2. SeqCalling~ Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if apprayi-iate. cDNA sequences from all ~ apples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is'included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.
3. PathCaIIing~ Technology:

The NOVX nucleic acid sequences are derived by laboratory screening of cDNA
library by the two-hybrid approach. cDNA fragments covering either the full length of the DNA sequence, or part of the sequence, or both, are sequenced. In silico prediction was based on sequences available in CuraGen Corporation's proprietary sequence databases or S in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.
The laboratory screening was performed using the methods summarized below:
cDNA libraries were derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then directionally cloned into the appropriate two-hybrid vector (Gal4-activation domain (Gal4-AD) fusion). Such cDNA
1 S libraries as well as commercially available cDNA libraries from Clontech (Palo Alto, CA) were then transferred from E.coli into a CuraGen Corporation proprietary yeast strain (disclosed in U. S. Patents 6,057,101 and 6,083,693, incorporated herein by reference in their entireties).
Gal4-binding doriiain (Gal4-BD) fusions of a CuraGen Corportion proprietary library of human sequences was used to screen multiple Gal4-AD fusion cDNA
libraries resulting in the selection of yeast hybrid diploids in each of :ul'uch tree GK,'~. ~A~? fusion contains an individual cDNA. Each sample was amplified using the polymerase chain reaction (PCR) using non-specific primers at the cDNA insert boundaries. Such PCR
product was sequenced; sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

Physical clone: the cDNA fragment derived by the screening procedure, covering the entire open reading frame is, as a recombinant DNA, cloned into pACT2 plasmid (CIontech) used to make the cDNA library. The recombinant plasmid is inserted into the host and selected by the yeast hybrid diploid generated during the screening procedure by the mating of both CuraGen Corporation proprietary yeast strains N106' and YULH (U. S.
Patents 6,057,101 and 6,083,693).
4. RACE: Techniques based on the polymerase chain reaction such as rapid amplification of cDNA ends (RACE), were used to isolate or complete the predicted sequence of the cDNA of the invention. Usually multiple clones were sequenced from one or more human samples to derive the sequences for fragments. Various human tissue samples from different donors were used for the RACE reaction. The sequences derived from these procedures were included in the SeqCalling Assembly process described in preceding paragraphs.
5. Exon Linking: The NOVX target sequences identified in the present invention I S were subjected to the exon linking process to confirm the sequence. PCR
primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by t~anslatPd homology of the predicted exons to closely related human sequences from other species. These primers were then employed in PGR amplification based on the following pool of human cDNAs:
adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain -hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector.
The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.
6. Physical Clone: Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail.
Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clones used for expression and screening purposes.
Example C: Quantitative expression analysis of clones in various cells and tissues The 'quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, Bell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on an Applied Biosystems ABI PRISM~ 7700 or an ABI PRISM~ 7900 HT Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel SD/SI (containing human tissues and cell lines with an emphasis on metabolic diseases), AI comprehensive~anel (containing normal tissue and samples from autoimmune diseases), Panel CNSD.O1 (containing central nervous system samples from normal and diseased brains) and CNS neurodegeneration~anel (containing samples from normal and Alzheimer's diseased brains).
17g RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA
contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, (3-actin and GAPDH). Normalized RNA (5 u1) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (Applied Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions.
In other cases, non-normalized RNA samples were converted to single strand eDNA
(sscDNA) using Superscript II (Invitrogen Corporation; Catalog No. 18064-147) and random hexamers according to the manufacturer's instructions. Reactions containing up to 10 ~g of total RNA were performed in a volume of 20 ~l and incubated for 60 minutes at 42 °C. This reaction can be scaled up to 50 ~.g of total RNA in a final volume of 100 ~1.
sscDNA samples are then normalized to reference nucleic acids as described previously, using 1X TaqMan~ Universal Master mix (Applied Biosystems; catalog No.
4324020), following the manufacturer's instructions.
Probes and primers were designed for each assay according to Applied Biosystems Primer Express Software package (version I for Apple Computer's Ma.,intosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (Tm) range = 58 °-60 °C, primer optimal Tm = 59 °C, maximum primer difference = 2 °C, probe does not have 5'G, probe Tm must be 10 °C greater than primer Tm, amplicon size 75bp to 100bp.
The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA).
Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900nM
each, and probe, 200nM.

PCR conditions: When working with RNA samples, normalized RNA from each tissue and each cell line was spotted in each well of either a 96 well or a 384-well PCR
plate (Applied Biosystems). PCR cocktails included either a single gene specific probe and primers set, or two multiplexed probe and primers sets (a set specific for the target clone S and another gene-specific set multiplexed with the target probe). PCR
reactions were set up using TaqMan~ One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No.
4313803) following manufacturer's instructions. Reverse transcription was performed at 48 °C for 30 minutes followed by amplification/PCR cycles as follows:
9S°C I O min, then 40 cycles of 95 °C for 1 S seconds, 60 °C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to ts~e rower ref delta C'''. 'TEL
percea relWi ae expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.
1 S When working with sscDNA samples, normalized sscDNA was used as described previously for RNA samples. PCR reactions containing one or two sets of probe and primers were set up as described previously, using 1X TaqMan~ Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions.
PCR amplification was performed as follows: 9S °C 10 min, then 40 cycles of 9S °C for 1 S
seconds, 60 °C for 1 minute. Results were analyzed and processed as described previously.
Panels 1,1.1,1.2, and 1.3D
The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control wells (genomic DNA
control and chemistry control) and 94 wells containing cDNA from various samples. The samples in these panels are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in these panels are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on these panels are comprised of samples derived from all major organ systems from single adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.
In the results for Panels 1, 1.l, 1.2 and 1.3D, the following abbreviations are used:
ca. = carcinoma, * = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, p1. eff = p1 effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.
General screening_panel v1.4 The plates for Panel 1.4 include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in Panel 1.4 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS
cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panel 1.4 are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panel 1.4 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose. Abbreviations are as described for Panels 1, 1.I, 1.2, and 1.3D.

Panels 2D and 2.2 The plates for Panels 2D and 2.2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins" obtained from noncancerous tissue just adjacent to the tumor.
These are termed normal adjacent tissues and are denoted "NAT" in the results below. The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologist at NDRI or CHTN). This analysis provides a gross histapathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue surrounding (i.e. immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen.
Panel3D
The plates of Panel 3D are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma of the tongue, breast cancer, prostate cancer, melanoma, epidermaid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D and 1.3D are of the most common cell lines used in the scientific literature.
Panels 4D, 4R, and 4.1D

Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, CA) and thymus and kidney (Clontech) was employed. Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc., Hayward, CA).
Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).
Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular.lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 1-Sng/ml, TNF alpha at approximately 5-l Ong/ml, IFN gamma at approximately 20-SOng/ml, IL-4 at approximately 5-lOng/ml, IL-9 at approximately 5-lOng/ml, IL-13 at approximately 5-l Ong/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1 % serum.
Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using Ficoll. LAK cells were prepared from these cells by culture in DMEM
5% FCS (Hyclone), 100~M non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO'SM
(Gibco), and l OmM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20ng/ml PMA and 1-2~.g/ml ionomycin, IL-12 at 5-l Ong/ml, IFN gamma at 20-SOng/ml and IL-18 at 5-l Ong/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100p.M non essential amino acids (Gibco), 1mM
sodium pyruvate (Gibco), mercaptoethanol S.SxlO'SM (Gibco), and l OmM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately S~.g/ml.
Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2x106cells/ml in DMEM 5% FCS (Hyclone), 100~,M
non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol (S.SxlO-SM) (Gibco), and l OmM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from I- 7 days for RNA preparation.
Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions.
Monocytes were differentiated into dendritic cells by culture in DMEM S% fetal calf serum (FCS) (Hyclone, Logan, UT), 100~.M non essential amino acids (Gibco), 1 mM
sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM (Gibco), and lOmM Hepes (Gibco), SOng/ml GMCSF and Sng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for S-7 days in DMEM 5% FCS (Hyclone), 100~M non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM (Gibco), lOmM
Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately SOng/ml.
Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with ' lipopolysaccharide (LPS) at 1 OOng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10~g/ml for 6 and 12-14 hours.
CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS
selection columns and a Vario Magnet according to the manufacturer's instructions.
CD45RA and CD45R0 CD4 lymphocytes were isolated by depleting mononuclear cells of CDB, CD56, CD14 and CD19 cells using CDB, CD56, CD14 and CD19 Miltenyi beads and positive selection. CD45R0 beads were then used to isolate the CD45R0 CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45R0 CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone),100pM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol S.Sx 10'5M
(Gibco), and l OmM Hepes (Gibco) and plated at 106cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with O.Spg/mI anti-CD28 (Pharmingen) and 3ug/ml anti-(OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA
preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), 100~.M non essential amino acids (Gibco), 1mM sodiumpynivate (Gibco), mercaptoethanol S.SxlO-SM (Gibco), and l OmM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days of the second expansion culture.
The isolated NK cells were cultured in DMEM 5% FCS (Hyclone),100pM non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM
(Gibco), and lOmM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 106 cells/ml in DMEM 5% FCS (Hyclone), 100~M non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco). mercaptoethanol S.SxlO'SM
(Gibco), and lOmM Hepes (Gibco). To activate the cells, we used PWM at Spg/ml or anti-(Pharmingen) at approximately lOpg/ml and IL-4 at 5-lOng/ml. Cells were harvested for RNA preparation at 24, 48 and 72 hours.
To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon plates were coated overnight with l Opg/ml anti-CD28 (Pharmingen) and 2~g/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, German Town, MD) were cultured at 105-106cells/ml in DMEM 5% FCS
(Hyclone), 100~M non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM (Gibco), lOmM Hepes (Gibco) and IL-2 (4ng/ml). IL-12 (Sng/ml) and anti-IL4 (1 pg/ml) were used to direct to Thl, while IL-4 (Sng/ml) and anti-IFN gamma (1 pg/ml) were used to direct to Th2 and IL-10 at Sng/ml was used to direct to Trl. After 4-5 days, the activated Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100p.M non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM
(Gibco), lOmM Hepes (Gibco) and IL-2 (lng/ml). Following this, the activated Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (lp,g/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in O.lmM dbcAMP at S Sx105cells/ml fox 8 days, changing the media every 3 days and adjusting the cell concentration to Sx105cells/ml. For the culture of these cells, we used DMEM
or RPMI (as recommended by the ATCC), with the addition of S% FCS (Hyclone), 100~M non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM
(Gibco), l OmM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 1 Ong/ml and ionomycin at 1 ~g/ml for 6 and 14 hours.
Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM S% FCS (Hyclone), 100~.M non essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), mercaptoethanol S.SxlO-SM (Gibco), and IOmM Hepes (Gibco). CCDl 106 cells were activated for 6 and 14 hours with 1S approximately S ng/ml TNF alpha and lng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: Sng/ml IL-4, Sng/ml IL-9, Sng/ml IL-13 and 2Sng/ml IFN gamma.
For these cell lines and blood cells, RNA was prepared by Iysing approximately I O~celIs/mI using TrizoI (Gibco BRL). Briefly, I/10 volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor.
The aqueous phase was removed and placed in a 1 Sml Falcon Tube. An equal volume of isopropanol was added and left at 20 °C overnight. The precipitated RNA
was spun down at 9,000 rpm for 1 S min in a Sorvall SS34 rotor and washed in 70% ethanol.
The pellet was 2S redissolved in 300.1 of RNAse-free water and 35.1 buffer (Promega) 5~.1 DTT, 7p.1 RNAsin and 8~.1 DNAse were added. The tube was incubated at 37 °C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of 100%
ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at -80 °C.
AI_comprehensive panel v1.0 The plates for AI comprehensive panel v1 .0 include two control wells and 89 test samples comprised of cDNA isolated from surgical and postmortem human tissues obtained from the Backus Hospital and Clinomics (Frederick, MD). Total RNA was extracted from tissue samples from the Backus Hospital in the Facility at CuraGen. Total RNA from other tissues was obtained from Clinomics.
Joint tissues including synovial fluid, synovium, bone and cartilage were obtained from patients undergoing total knee or hip replacement surgery at the Backus Hospital.
Tissue samples were immediately snap frozen in liquid nitrogen to ensure that isolated RNA was of optimal quality and not degraded. Additional samples of osteoarthritis and rheumatoid arthritis joint tissues were obtained from Clinomics. Normal control tissues were supplied by Clinomics and were obtained during autopsy of trauma victims.
Surgical specimens of psoriatic tissues and adjacent matched tissues were provided as total RNA by Clinomics. Two male and two female patients were selected between the ages of 25 and ,47. None of the patients were taking prescription drugs at the time samples were isolated.
Surgical specimens of diseased colon from patients with ulcerative colitis and Crohns disease and adjacent matched tissues were obtained from Clinomics.
Bowel tissue from three female and three male Crohn's patients between the ages of 41-69 were used.
Two patients were not on prescription medication while the others were taking dexamethasone, phenobarbital, or tylenol. Ulcerative colitis tissue was from three male and four female patients. Four of tile patients were taking leb vid ~~u w~ w;.re or~
phenobarbital.
Total RNA from post mortem lung tissue from trauma victims with no disease or with emphysema, asthma or COPD was purchased from Clinomics. Emphysema patients ranged in age from 40-70 and all were smokers, this age range was chosen to focus on patients with cigarette-linked emphysema and to avoid those patients with alpha-lanti-trypsin deficiencies. Asthma patients ranged in age from 36-75, and excluded smokers to prevent those patients that could also have COPD. COPD patients ranged in age from 35-80 and included both smokers and non-smokers. Most patients were taking corticosteroids, and bronchodilators.

In the labels employed to identify tissues in the AI comprehensive panel v1.0 panel, the following abbreviations are used:
AI = Autoimmunity Syn = Synovial Normal = No apparent disease Rep22 /Rep20 = individual patients RA = Rheumatoid arthritis Backus = From Backus Hospital OA = Osteoarthritis (SS) (BA) (MF) = Individual patients Adj = Adjacent tissue Match control = adjacent tissues -M = Male -F = Female COPD = Chronic obstructive pulmonary disease Panels SD and SI
The plates for Panel SD and SI include two control wells and a variety of cDNAs isolated from human tissues and cell lines with an emphasis on metabolic diseases.
Metabolic tissues were obtained from patients enrolled in the Gestational Diabetes study.
Cells were obtained during different stages in the differentiation of adipocytes from human mesenchymal stem cells. Human pancreatic islets were also obtained.
In the Gestational Diabetes study subjects are young (18 - 40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective) Caesarean section. After delivery of the infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<1 cc) of the exposed metaboirc tissues during the closure of each surgical level. The biopsy material was rinsed in sterile saline, blotted and fast frozen within 5 minutes from the time of removal. The tissue was then flash frozen in liquid nitrogen and stored, individually, in sterile screw top tubes and kept on dry ice for shipment to or to be picked up by CuraGen. The metabolic tissues of interest include uterine wall (smooth muscle), visceral adipose, skeletal muscle (rectos) and subcutaneous adipose. Patient descriptions are as follows:
Patient 2 Diabetic Hispanic, overweight, not on insulin Patient ?-9 Nondiabetic Caucasian and obese (BMI>30) Patient 10 Diabetic Hispanic, overweight, on insulin Patient 11 Nondiabetic African American and overweight Patient 12 Diabetic Hispanic on insulin Adipocyte differentiation was induced in donor progenitor cells obtained from Osirus (a division of CloneticsBioWhittaker) in triplicate, except for Donor 3U which had only two replicates. Scientists at Clonetics isolated, grew and differentiated human mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol found in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells Science Apr 2 1999: 143-147. Clonetics provided Trizol lysates or frozen pellets suitable for mRNA isolation and ds cDNA production. A general description of each donor is as follows:
Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated Adipose I O Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated Donor 2 and 3 AD: Adipose, Adipose Differentiated Human cell lines were generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups:
kidney proximal convoluted tubule, uterine smooth muscle cells, small intestine, Liver HepG2 cancer cells, heart primary stromal cells, and adrenal cortical adenoma cells. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. All samples were processed at CuraGen to produce single stranded cDNA.
Panel SI contains all samples previously described with the addition of pancreatic islets from a 58 year old female patient obtained from the Diabetes Research Institute at the University of Miami School of Medicine. Islet tissue was processed to total RNA at an outside source and delivered to Curarsen for addition to panel 5I.
In the labels employed to identify tissues in the SD and SI panels, the following abbreviations are used:
GO Adipose = Greater Omentum Adipose SK = Skeletal Muscle UT = Uterus PL = Placenta AD = Adipose Differentiated AM = Adipose Midway Differentiated U = Undifferentiated Stem Cells Panel CNSD.Ol The plates for Panel CNSD.O1 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains two brains from each of the following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy, Depression, and "Normal controls".
Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases;
e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration of the substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.
In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:
PSP = Progressive supranuclear palsy - Sub Nigra = Substantia nigra . -Glob PaIladus= Globus palladus Temp Pole = Temporal pole Cing Gyr = Cingulate gyrus BA 4 = Brodman Area 4 Panel CNS Neurodegeneration V1.0 The plates for Panel CNS Neurodegeneration V1.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System).
Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains six brains from Alzheimer's disease (AD) patients, and eight brains from "Normal controls" who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0 = no evidence of plaques, 3 = severe AD senile plaque load). Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Brodman Area 21), parietal cortex (Brodman area 7), and occipital cortex (Brodman area 17).
These reg o_n_s were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal Loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages of the disease; the occipital cortex is.
spared in AD and therefore acts as a "control" region within AD patients. Not all brain regions are represented in all cases.
In the labels employed to identify tissues in the CNS Neurodegeneration V 1.0 panel, the following abbreviations are used:
AD = Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy Control = Control brains; patient not demented, showing no neuropathology Control (Path) = Control brains; pateint not demented but showing sever AD-like pathology SupTemporal Ctx = Superior Temporal Cortex Inf Temporal Ctx = Inferior Temporal Cortex A. CG100104-Ol: fibronectin-malate dehydrogenase Expression of gene CG100104-01 was assessed using the primer-probe set Ag4162, described in Table AA. Results of the RTQ-PCR runs are shown in Tables AB and AC.

Table AA. Probe Name Ag4162 Start SEQ ID

PrimersSequences Length _ P_o__sitionNo ~

Forward5~-cctcagttatgctcctgtctgt-3~ 22 _11s 93 _ Probe TES'-ttcatcttcacctcagagcggaactg-3'-26 14S 94 Reverse5~-cagttccgctcatctttgtaag-3~ 22 188 95 Table AB. General screening~anel v1.4 Rel. Exp.(!) Rel. Exp.(e) Tissue Name Ag4162, Run Tissue Name Ag4162, Run Adipose 0.0 Renal ca. TK-10 0.2 MeIanoma* 0.0 Bladder 0.5 ~

_ .._. . ..... .. _ . . . __ _. .._.__._..
A).T .. ... . ._ .._ _. _ _ _ . .. . ..
Hs688( . . . ..... . ...... ._..... . .
.. __.. . .. .
_ Melanoma* Gastric ca. (liver 0'0 met.) 0.2 Hs688(B).T CI-N87 Melanoma* M14 ~ 0.0 Gastric ca. KATO 0.0 III

Melanoma* 0.0 Colon ca. SW-948 0.0 LOXIMVI

Melanoma* SK- 0.1 Colon ca. SW480 O.s ~

MEL-5 .. . . _ ._ . _. _ . .. ._ _ . . _ . . . _. .
_ . .. _ . ........ ... . _. . ._.
. ._. . _ . .

Squamous cell Colon ca.* (SW480 0'0 0.0 carcinoma SCC-4 met) SW620 Testis Pool 100.0 Colon ca. HT29 0.2 Prostate ca.* 0.0 Colon ca. HCT-116 0.0 (bone met) PC-3 Prostate Pool 0.2 Colon ca. CaCo-2 0.6 Placenta 0.0 Colon cancer tissue0.0 Uterus Pool 0.0 Colon ca. SW1116 0.0 Ovarian ca. 0.4 Colon ca. Colo-20s 0.0 Ovarian ca. SK- 0.0 Colon ca. SW-48 0.0 ~

OV-3 _ _ _ Ovarian ca . 0.0 Colon Pool 0.3 Ovarian ca. p. l Small Intestine 0.0 Pool ovcAR-s Ovarian ca.

IGROV-1 0.0 Stomach Pool 0.0 Ovarian ca. 3.4 Bone Marrow Pool 0.0 OVCAR-8 ..~."",.,.~.....-,...,-,_,~..,..~, ~,~. .~.",.~..~..,",W".-..~.

Ovary 0.0 ~ Fetal Heart 0.0 Breast_ca. MCF-7 0.5 Heart Pool 0.0 _ ' Breast ca. MDA- 0.0 Lymph Node Pool 0.2 Breast ca. BT 549 0.0 Fetal Skeletal Muscle0.1 Breast ca. T47D 0.0 Skeletal Muscle 0.7 Pool Breast ca. MDA-N 0.0 Spleen Pool 0.0 Breast Pool 0.2 Thymus Pool 0.2 CNS cancer Trachea 0.3 0 (glio/astro) U87-MG.

CNS cancer Lung 0.2 0.3 (glio/astro) U-118-MG

CNS cancer Fetal Lung 0.1 ~ 0.0 (_n_~prn~m__Ptl ~ $T_~ N-A C ~
~

Lung ca. NCI-N417 0.0 cancer (astro) SF- 0.0 ~ 9S

' Lung ca. LX-1 0.0 CNS cancer (astro) p.0 Lung ca. NCI-H146 p.7 CNS cancer (glio) 0.0 CNS cancer (glio) Lung ca. SHP-77 0.5 SF 0.0 ~ ~

.. _ .... _.._.__ ..._. _.._.._...295 _. .._.. .
_ . ... .. ..... ._. . _.._.._.. ..._.... .....__ ..
._ .. .. .. ._. . ....___ .... .. . .. .
._.... ... ..
... .. ...
._..._.__ . .. ..

g 0.0 , _ 0.0 Lun _ca. A549 ( ~
yg ) Brain Am data Pool Lung ca. NCI-H526_0.0 Brain (cerebellum) 0.0 Lung ca. NCI-H23 0.4 _ Brain (fetal) _ 0.0 _ ___ Lung ca. NCI-H460 0.2 Brain (Hippocampus)03 Pool .

Lung ca. HOP-62 0.0 Cerebral Cortex 0.8 ._ -_.-.. .~~ Pool __._.
....r...,. _ __..._._.___..
_..._.._._.~""~.

Brain (Substantia Lung ca. NCI-H522 0.4 0.0 n igra) Pool Liver 0.0 Brain (Thalamus) 0.0 Pool Fetal Liver 0.0 Brain (whole) 0.0 Liver ca. HepG2 0.0 Spinal Cord Pool 0.0 Kidney Pool 0.4 Adrenal Gland 0.1 Fetal Kidney 0.0 Pituitary gland 0.0 Pool Renal ca. 786-0 0.0 Salivary Gland 0.0 Renal ca. A498 0.0 Thyroid (female) 0.0 Pancreatic ca.
Renal ca. ACHN 0.2 0 CAPAN2 .

Renal ca. U0-31 0.0 Pancreas Pool 0.2 Table AC. Panel 4.1 D

Rel. Exp.(%)~~ Rel. Exp.(f) Tissue Name Ag4162, Run Tissue Name Ag4162, Run Secondary Thl 0.0 HUVEC IL-lbeta 0.0 act Secondary Th2 0.0 HUVEC IFN gamma 0.0 act Secondary Trl 0.0 SEC TNF alpha + 0.0 act IFN gamma Secondary Thl 0.0 HUVEC TNF alpha + 0.0 rest IL4 Secondary Th2 0.0 HUVEC IL-11 0.0 rest Lung Microvascular Secondary Trl ~.0 EC 0.0 rest ~

. ._._ _. .. .._._.__ _.none.__..__.____...Y
. _._ __..__._ __ ._. _ _._ -_. _ _ _. _. _... _ ._ Primary Thl act 0.0 Leg Microvascular _ EC 0.0 TNFalpha + IL-1 beta Primary Th2 act 0.0 Microvascular Dermal0.0 ~

EC none Primary Trl act 0.0 Microsvasular Dermal0.0 EC

TNFalpha + IL-lbeta Bronchial epithelium Primary Thl rest 0.0 ~ 0.0 _ . TNFalpha + IL 1 beta_ Primary Th2 rest 0.0 small airway epithelium0.0 none Primary Trl rest 0.0 Small airway epithelium0.0 TNFalpha + IL-1 beta 0.0 Coronery artery SMC 0.0 rest lymphocyte act CD45R0 CD4 Coronery artery SMC
0'0 0 lymphocyte act , _ TNFalpha + IL-lbeta .
_ ~ ~. .

CD8 lymphocyte_ 0.0 Astrocytes rest 0.0 act Secondary CD8 Astrocytes TNFalpha 0 + 0 0 ~ 0 lymphocyte rest ' ~IL-lbeta .

Secondary CD8 0,0 KU-812 (Basophil) 0.0 rest lymphocyte act CD4 lymphocyte 0.0 KU-812 (Basophil) 0.0 none PMA/ionomycin try Thl/Th2/Trl CCD1106 anti-_ 0'0 ~(Keratinocytes) 0.0 CD95 CHl 1 __ _._. none __ _ . . .. ___ _ -. . _ __ .
~

CCD

LAK cells rest 0.0 (Keratinocytes) 0.0 TNFalpha + IL-lbeta LAK cells IL-2 0.0 Liver cirrhosis 0.5 LAK cells IL-2+IL-120.0 CI-H292 none 0.0 LAK cells IL-2+IFN

0.0 CI-H292 IL-4 0.0 gamma LAK cells IL-2+ 0.0 CI-H292 IL-9 0.0 LAK cells 0.0 NCI-H292 IL-13 ~ 0 PMA/ionomycin .

NK Cells IL-2 rest 0.0 NCI-H292 IFN gamma 0.0 Two Way MLR 3 day _ 0.0 HPAEC none 0.0 Two Way MLR 5 day 0.0 HPAEC TNF alpha + IL- 0.0 1 beta Two Way MLR 7 day 0.0 Lung fibroblast none 0.0 PBMC rest 0.0 Leg fibroblast TNF 0 alpha + IL-1 beta .

PBMC PWM 0.0 Lung fibroblast IL-4 0.0 PBMC PHA-L 0.0 Lung fibroblast IL-9 0.0 Ramos (B cell) none0.0 Lung fibroblast IL-13 0.6 Ramos (B cell) 0.6 Lung fibroblast IFN 0.0 .

ionomycin . .. gamma ..

De~rira' fi br Vblast B lymphocytes PWM 0.0 0.0 CCD1070 rest ~

B lymphocytes CD40L Dermal fibroblast and IL-4 0'0 CCD1070 TNF alpha 0.6 EOL-l dbcAMP 0.0 Dermal fibroblast 0 CCD1070IL-1 beta .

EOL-1 dbcAMP 0.0 Dermal fibroblast IFN 0 PMAlionomycin....__.___.._______._._.___..._..._.~ga _.... ___._...
_._..~. _ ..... _._..__._.... __._ ._ _ ~.._. _ _-.___ _.. _ _ . _.

Dendritic cells 0.0 _ _ Dermal fibroblast 0.0 none_ IL-4 _ Dendritic cells 0.0 _ __ Dermal Fibroblasts 1.1 LPS rest Dendritic cells 0.0 Neutrophils TNFa+LPS 1.4 anti-Monocytes rest 0.0 Neutrophils rest 1.0 Monocytes LPS 0.0 Colon 2.2 Macrophages rest 0.0 Lung _11.3 ~

Macrophages LPS 0.0 Thymus 10.7~~

HUVEC none 0.0 Kidney 100.0 HUVEC starved 0.0 General screening_panel v1.4 Summary: Ag4162 Highest expression of the CG100104-O1 gene is detected exclusively in testis (CT=28.5). Therefore, expression of this gene could be used to distinguish testis sample from other samples used in this panel.
In addition, therapeutic modulation of this gene product could be useful in treatment of testis related disorders such fertility and hypogonadism.

In addition, low expression of this gene is also detected in Ovarian cancer OVCAR-8 cell line (CT--33.4). Therefore, therapeutic modulation of this protein product may be useful in the treatment of ovarian cancer.
Panel 4.1D Summary: Ag4162 Highest expression of the CG100104-O1 gene is detected in kidney (CT=29.9). Expression of this gene is exclusively seen in normal lung, thymus and kidney. Thus expression of this gene could be used to distinguish these tissue samples from other samples in this panel. In addition, therapeutic modulation of this gene product could be beneficial in the treatment of inflammatory or autoimmune diseases that affect lung and kidney.
B. CG56785-Ol: GTP:AMP PHOSPHOTRANSFERASE
MITOCHONDRIAL
Expression of gene CG56785-O1 was assessed using the primer-probe set Ag3036, described in Table BA. Results of the RTQ-PCR runs are shown in Tables BB, BC
and BD.
Table BA. Probe Name Ag3036 _..~,.....,......._,..,.~..,..-.~..-.-...~..
Start SEQ ID
rimersSequences Length positionNo Forward5'-accaatggccaagtctacaac-3' 21 427 96 PI'ObeTET-5'-attggattcaaccctcccacaactgt-3'-' Reyerse5'-gtttateatcctcacgetgaat-3' 22 505 98 Table BB. CNS neurodegeneration v1.0 -_ Rel. Ezp.(%) Ag3036,~ Rel. Exln_l/..1 Tissue Name Run 211012102 Tissue Nam A o~n36.
e Run 211012102 Control (Path) AD 1 Hippo 3.3 3 5.8 Temporal Ctx Control (Path) AD 2 Hippo 4.9 4 11.0 _ _ Temporal Ctx AD 3 Hippo 0.0 AD 1 Occipital_Ctx11.9 AD 4 Hippo 2.9 AD 2 Occipital__ Ctx 0.0 (Missing) AD 5 Hippo 100.0 AD 3 Occipital0.0 Ctx AD 6 Hippo 6.9 AD 4 Occipital9.2 Ctx Control 2 4.2 AD S Occipital3.3 Hippo Ctx Control 4 3.3 AD 6 Occipital1.9 Hippo Ctx Control (Path)7.9 Control 1 Occipital0.0 Hippo AD 1 Temporal ~ 5.0 ~~ ntrol 2~~Occipital ~...~.....-.., i ~...~.-... g ~ ~ C g o C C .
~

AD 2 Temporal 18.2 Control 3 Occipital ~ 5 Ctx Ctx .

AD 3 Temporal 3.3 Control 4 Occipital 0 Ctx Ctx .

AD 4 Temporal 11.0 Control (Path) 1 Sg C~_ .__... ..._......._.._....___ _ _ _ _ . .... occipital .
_ _.._ _ ~.. ... Ctx. .._. .... . .__..._._ .___.__....
_ _.~ _.. . ... .._..._ ._ .. .._ __ AD 5 Inf Temporal Control (Path) 2 ' 6'5 3 Cbc Occipital Ctx .

AD 5 Sup Control (Path) 3 40.3 4 Temporal Ctx Occipital Ctx .

AD 6 Inf Temporal Control (Path) 4 10.2 4 C~ Occipital Ctx .

AD 6 Sup Control 1 Parietal ~ 9'3 ~ 0 _. C~ .
Temporal_ Ctx Control 1 _ 0.0 Control 2 Parietal 32 Temporal Ctx C~ ~ .

Control 2 Control 3 Parietal 3' I 8 Temporal Ctx C~ .

Control 3 Control (Path) 1 2'2 43 Temporal Ctx Parietal Ctx .

Control 3 Control (Path) 2 8'S 5 Temporal Ctx ._ _ _ . Parietal Ctx .
_ __...._ _. ._ _. ...... _ . . .._ .. . . _ .._.
_ _. .... .... ...... . _ . . ...._ .. . ...... ..._.

Control (Path) Control (Path) 3 1 50'7 1 Temporal Ctx Parietal Ctae .

Control (Path) Control (Path) 4 Temporal Ctx ' Parietal Ctx 14.4 Table BC. Panel I.3D
Rel. Exp.(%) Rel. Ezp.(!) Tissue Name Ag3036, Run Tissue Name Ag3036, Run 167962459 ~ 167962459 Liver adenocarcinoma0.0 Kidney (fetal) 8.1 Pancreas 0.0 Renal ca. 786-0 3.2 Pancreatic ca. 0.0 Renal ca. A498 0 2_.. ...... _ . .
_ _ .

_ .__.__......._.......__._....... . ... ..
Adrenal gland 0.0 ... _._ . 0.0 _..._........___..._.._.. Renal ca. RXF
__. _....__... 393 __ _.._ .__.____ ._. _ ~

__.. _.._ _ _ _ __ Thyroid 0.0 .._ 8.2 -.Renal ca. ACHN
~

Salivary._gland 0.0 _._ _ Renal c_a. U0-310.0 ~

Pituitary gland 0.0 __ Renal ca. 0.0 TK-I O

Brain (fetal) 27.9 _ Liver _ 21.9 _ Brain (whole) 22.4 Liver (fetal) 38.4 Brain (amygdala)~._ ~ 38.7 ' Liver ca. ~ ,~,~"""~"",_0_.0 ....... ...."~,~.v,........._..............._.

(hepatoblast) HepG2 Brain (cerebellum) 38.4 Lung 17.1 Brain (hippocampus) 4.7 Lung (fetal) 3.4 Brain (substantia 4,2 LX g ca. (small 0.0 nigra) cell) Brain (thalamus) 19.8 Leg ca. (small p.0 cell) Lung ca. (s.cell Cerebral Cortex 13.5 var.) 0 _ _._ _ . . ....... SHP-77_.
. . _ .. . _._ _ _. .. .. .. ._.._ ... _ ... _ Spinal cord 0.0 Leg ca. (large 0.0 cell)NCI-H460 glio/astro U87-MG 0.0 Leg ca. (non-sm. 0.0 cell) A549 glio/astro U-118-MG 0.0 Leg ca. (non-s.cell)0.0 Lung ca. (non-s.cell) astrocytoma SW1783 ~ .0 . . _. ... ._._...~_~...HOP.-62_ .._._. ......_. ._..._..._.._.
_ . . _ . ..._. ....._ _._~
.. .._._ _. __ . . _ _ .
neuro*; met SK-N-AS 0.0 Leg ca. (non-s.cl)0.0 astrocytoma SF-539 0.0 Leg ca. (squam.) 0.0 ~

astrocytoma SNB-75 0.0 Leg ca. (squam.) 0.0 glioma SNB-19 0.0 Mammary gland 0.0 Breast ca.* (pl.ef) glioma U251 0.0 0.0 .. _ . _ . . .._ _. MCF-7 ' _._._..._. _ . .. _. _ _ . .. ..__ . . _ . _._ _. . .
_.

Breast ca.* (pl.efj glioma SF-295 0.0 0.0 ~

Heart (fetal) 0.0 Breast ca.* (nl.efl~,0 Heart 0.0 Breast ca. BT-5490.0 Skeletal muscle (fetal)2.7 Breast ca. MDA 0.0 N

Skeletal muscle 4.1 Ovary 0.0 Ovarian ca.
Bone marrow 100.0 0 OVCAR-3 .

Ovarian ca.
Thymus 0.0 0.0 OVCAR-4 __ _ .

Ovarian ca.
Spleen 31.2 0.0 Lymph node 0.0 ' ~ 0.0 OVC~ 8 Colorectal 0.0 Ovarian ca. IGROV-0 .

Stomach 0.0 Ovanan ca.* 0.0 (ascites) SK-OV-3 Small intestine 0.0 Uterus 6.2 Colon ca. SW480 0.0 Placenta 0.0 Colon ca.* e 0.0 Prostat 0.0 SW620(SW480 met) Colon ca. HT29 0.0 Prostate ca.* (bone 0.0 met)PC-3 Colon ca. HCT-116 0.0 Testis 0.0 Colon ca. CaCo-2 0.0 Melanoma 0.0 _. _._.._ ... ..__ _ ___ Hs688(A).T
_ . _ . _ _._ _ .. ._ __... .__... _. _...._._......~.._.. _..._.
__ . ... . ... . . . _ Colon ca. Melanoma* (met) tissue(OD03866) ' Hs688(B).T .

Colon ca. HCC-2998 0.0 6 elanoma UACC- ~ 0.0 .

Gastric ca.* (liver 0 Melanoma M14 0 met) 0 0 NCI-N87 . .

Melanoma LOX
Bladder 0.0 0 _. . __. . _ ICI .
_ _._. _._ . _ .._ __ _ _.
_.. _ Trachea 3.5 Melanoma* (met) 0 SK-MEL-5 .

Kidney 0.0 Adipose ~9.8 Table BD. Panel 4D
Rel. Exp.(%) Rel. Exp.(%) Tissue Name Ag3036, Run Tissue Name Ag3036, Run Secondary Thl 0.0 HUVEC IL-lbeta 0.0 act Secondary Th2 2.5 HUVEC IFN gamma 0.0 act ~

Secondary Trl 0.0 SEC TNF alpha + 0.0 act IFN gamma Secondary Thl 0.0 HUVEC TNF alpha 0.0 rest + IL4 Secondary Th2 0.0 HUVEC IL-11 0.0 rest Lung Microvascular Secondary Trl 1.7 EC 0.0 rest .

_ . . . _..... _. . _ _ ._. none. ... __ . _ .__. _. . _ _._._. _ ._ _ _ Primary Thl act 0.0 Leg Microvascular 0.0 EC

TNFalpha+ IL-lbeta.

Primary Th2 act 0.0 Microvascular Dermal0.0 EC none Primary Trl act 4.4 Microsvasular Dermal0,0 EC

TNFalpha + IL-lbeta Primary Thl rest0.0 Bronchial epithelium0.0 TNFalpha + ILlbeta Primary Th2 rest 0.0~ Small airway~.epithelium none Primary Trl rest . 0.0 Small airway epithelium 0 .
TNFalpha + IL-1 beta 2.1 Coronery artery SMC rest 0.0 lymphocyte act CD45R0 CD4 Coronery artery SMC

lymphocyte act ' TNFalpha + IL-lbeta 0.0 . _. _ _ ...._.... . . . ._ . __.. _._...
. _ _ _ ...._ ...___..
... .._. . _.
_ CD8 y._ .. O-0 lymphoc _ 1.'~ . ..
a ac_,_.. ... Astr'ocytes _ .._ . _ _.... rest . _. _.. _. ~ . .__. __. ._ __. _.. __ _ _.
_ t . .. .._ _ .__ .__ _ .
Secondary CD8 0 _ 0 Astrocytes TNFalpha +

lymphocyte rest ' IL-lbeta 0.0 Secondary CD8 0.0 KU-812 (Basophil) rest 0.0 lymphocyte act KU-812 (Basophil) CD4 lymphocyte 0.0 0.0 none p~ionomycin 2 Thl/Th2/TrI CCD1106 anti-ry .

0.0 0 0 CD95 CH11 eratinoc es none ' ._ .. ._..._... _. .___.....
~.................._._........~.........~..........._.._..._......_.._.._._.._.
_ ......_ _.. .... ... _.._._..._.... . . _. . ___._. _._..._ .
..._. ..._....._ _.._.... ... . .__.
.... ... ......

LAK cells rest 1.9 (Keratinocytes) 0.0 TNFalpha + IL-1 beta LAK cells IL-2 0.0 Liver cirrhosis 1 S.5 LAK cells IL-2+IL-120.0 Lupus kidney 0.0 LAK cells IL-2+IFN0.0 NCI-H292 none . 0.0 gamma LAK cells IL-2+ 0.0 NCI-H292 IL-4 0.0 LAK cells 0.0 CI-H292 IL-9 0 1' PMA/ionomyc_in .
. _ _ .
_.

NK Cells 0.0 NCI-H292 IL-2 rest IL-13 . 0.0 .
..

Two Way MLR 3 3.5 NCI-H292 IFN
day __ __. . .._ gamma _._____....... . __.__. . 0.0 ._._.. ... .... .._._.._..... _._ _._. . ...
. .__._ . __ . ..__ ._._. .__. _.. . .__..
. _._. ___ .. _ .. - __ ....__ Two Way MLR 5 0.0 HPAEC none day Two Way MLR 7 2.2 HPAEC TNF alpha + IL- 0.0 day 1 beta PBMC rest 12.3 Lung fibroblast none 0.0 PBMC PWM 0.0 Leg fibroblast TNF 0 .
alpha + IL-1 beta PBMC PHA-L 3.0 Lung fibroblast IL-4 0.0 Ramos (B cell) 0.0 Lung fibroblast IL-9 0.0 none Ramos (B cell) 0.0 Lung fibroblast IL-13 0.0 ionomycin Lung fibroblast IFN
B lymphocytes 11.0 0.0 PWM

._ . . _. _ . ga _ _ _. .. _ . __ . . __ B lymphocytes Dermal fibroblast and IL-4 1$'3 CCD1070 rest 0.0 EOL-1 dbcAMP 0.0 Dermal fibroblast 1.1 _ "~,~,-"""~",~" C_CD_107_0 TNF
alpha EOL-1 dbcAMP Dermal fibroblast PMAiionomycin 0'0 CCD1070IL-1 beta 0.0 Dendritic cells 0.0 g~~ fibroblast IFN p.0 none Dendritic cells 0.0 Dermal fibroblast 0.0 Dendritic cells anti-0,0 IBD Colitis 2 0.0 Monocytes rest 100.0 IBD Crohn's 0.0 Monocytes LPS 14.0 Colon 0.0 Macrophages rest 0.0 Lung 10.7 Macrophages LPS 2.3 Thymus 1.1 HI1VEC none 0.0 Kidney 3.1 HUVEC starved 0.0 CNS ~eurodegeneration v1.0 Summary: Ag3036 T'nis panel does not show differential expression of the CG56785-Ol gene in Alzheimer's disease. However, this expression profile does show the presence of this gene in the brain. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.
Panel 1.3D Summary: Ag3036 Expression of the CG56785-Ol gene is exclusive to bone marrow (CT=34.6). This gene encodes a putative member of the adenylate kinase family, which has been shown to be down-regulated in various blood disorders (Walter HD, Klin Wochenschr 1978 May 15;56(10):483-91). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker of bone marrow and red blood cells. Furthermore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of blood disorders and leukemias.
Panel 4D Summary: Ag3036 Expression of the CG56785-O1 gene is exclusive to resting monocytes (CT=33.3). This expression is in agreement with expression in Panel 1.3D. The expression of this gene in resting cells of this lineage suggests that the protein encoded by this transcript may be involved in normal immunological processes associated with immune homeostasis.
C. CG56914-O1: Thrombospondin DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:

Claims (50)

What is claimed is:

1. An isolated polypeptide comprising the mature form of an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 46.

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 46.

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 46.

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 46.

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 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, 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 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 46 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 46.

21. The nucleic acid molecule of claim 20, wherein the nucleic acid molecule is naturally occuring.

22. A nucleic acid molecule, wherein the nucleic acid molecule differs 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 46.

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 46.

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 46.

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 46, 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. The antibody of claim 29, wherein the antibody is a fully human antibody.

33. The antibody of claim 29, wherein the dissociation constant for the binding of the polypeptide to the antibody is less than 1 × 10-9 M.

34. The antibody of claim 29, wherein the antibody neutralizes an activity of the polypeptide.

35. A method of treating or preventing a NOVX-associated disorder, the method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 29 in an amount sufficient to treat or prevent the pathology in the subject.

36. The method of claim 35, wherein the subject is human.

37. 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;
(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.

38. The method of claim 37 wherein presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type.

39. The method of claim 38 wherein the cell or tissue type is cancerous.

40. 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.

41. 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 46.

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.

46. 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 46.

47. The method of claim 46 wherein the cell is a bacterial cell.

48. The method of claim 46 wherein the cell is an insect cell.

49. The method of claim 46 wherein the cell is a yeast cell.

50. The method of claim 46 wherein the cell is a mammalian cell.