CA2460653A1 - Therapeutic polypeptides, nucleic acids encoding same, and methods of use - Google Patents

Abstract

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

Description

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

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 xesults 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 ~5 way of nonlimiting example induction of cell or tissue proliferation, suppression of growth or proliferation, induction of differentiation or maturation of a cell or tissue, and suppression of differentiation or maturation of a cell or tissue.
Many pathological conditions involve dysregulation of expression of important efFector proteins. In certain classes of pathologies the dysregulation is manifested as diminished or suppressed level of synthesis and secretion of protein effectors. In other classes of pathologies the dysregulation is manifested as increased or up-regulated level of synthesis and secretion of protein effectors. In a clinical setting a subject may be suspected of suffering from a condition brought on by altered or mis-regulated levels of a protein effector of interest. Therefore there is a need to assay for the level of the protein effector of interest in a biological sample from such a subject, and to compare the level with that characteristic of a nonpathological condition. There also is a need to provide the protein effector as a product of manufacture. Administration of the effector to a subject in need thereof is useful in treatment of the pathological condition. Accordingly, there is a need for a method of treatment of a pathological condition brought on by a diminished or suppressed levels of the protein efFector of interest. In addition, there is a need for a method of treatment of a pathological condition brought on by a increased or up-regulated levels of the protein efFector of interest.
Antibodies are multichain proteins that bind specifically to a given antigen, and bind poorly, or not at all, to substances deemed not to be cognate antigens.
Antibodies are comprised of two short chains termed light chains and two long chains termed heavy chains.
These chains are constituted of immunoglobulin domains, of which generally there are two classes: one variable domain per chain, one constant domain in light chains, and three or more constant domains in heavy chains. The antigen-specific portion of the immunoglobulin molecules resides in the variable domains; the variable domains of one light chain and one heavy chain associate with each other to generate the antigen-binding moiety.
Antibodies that bind immunospecifically to a cognate or target antigen bind with high affinities.
Accordingly, they are useful in assaying specifically for the presence of the antigen in a sample. In addition, they have the potential of inactivating the activity of the antigen.
Therefore there is a need to assay for the level of a protein effector of interest in a biological sample from such a subject, and to compare this level with that characteristic of a nonpathological condition. In particular, there is a need for such an assay based on the use of an antibody that binds immunospecifically to the antigen. There further is a need to inhibit the activity of the protein effector in cases where a pathological condition arises from elevated or excessive levels of the effector based on the use of an antibody that binds immunospecifically to the effector. Thus, there is a need for the antibody as a product of manufacture. There further is a need for a method of treatment of a pathological condition brought on by an elevated or excessive level of the protein effector of interest based on administering the antibody to the subject.

SUn~VIARY OF THE INVENTION
The invention is based in part upon the discovery of isolated polypeptides including amino acid sequences selected from mature forms of the amino acid sequences selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOVl, 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.
The invention also is based in part upon variants of a mature form of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed. In another embodiment, the invention includes the amino acid sequences selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141. In another embodiment, the invention also comprises variants of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141 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 involves fragments of any of the mature forms of the amino acid sequences selected from the group consisting of SEQ ID
N0:2n, wherein n is an integer between 1 and 141, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to 15% of the residues are changed.
In another embodiment, the invention encompasses polypeptides that are naturally occurring allelic variants of the sequence selected from the group consisting of SEQ ID
N0:2n, wherein n is an integer between 1 and 141. These allelic variants include amino acid sequences that are the translations of nucleic acid sequences differing by a single nucleotide from nucleic acid sequences selected from the group consisting of SEQ ID NOS:
2n-1, wherein n is an integer between 1 and 141. The variant polypeptide where any amino acid changed in the chosen sequence is changed to provide a conservative substitution.
In another embodiment, the invention comprises a pharmaceutical composition involving a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141 and a pharmaceutically acceptable carrier. In another embodinnent, the invention involves a kit, including, in one or more containers, this pharmaceutical composition.
In another embodiment, the invention includes the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease being selected from a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ TD N0:2n, wherein n is an integer between 1 and 141 wherein said therapeutic is the polypeptide selected from this group.
In another embodiment, the invention comprises a method for determining the presence or amount of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, whereili n is an integer between 1 and 141 in a sample, the method involving providing the sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the polypeptide, thereby determining the presence or amount of polypeptide in the sample.
In another embodiment, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a polypeptide with an amino acid sequence selected from the group consisting of SEQ 1D N0:2n, wherein n is an integer between 1 and 141 in a first mammalian subject, the method involving measuring the level of expression of the polypeptide in a sample from the first mammalian subject;
and comparing the amount of the polypeptide in this sample to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, the disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
In another embodiment, the invention involves a method of identifying an agent that binds to a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141, the method including introducing the polypeptide to the agent; and determining whether the agent binds to the polypeptide. The agent could be a cellular receptor or a downstream effector.
In another embodiment, the invention involves a method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to abexrant expression or aberrant physiological interactions of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141, the method including providing a cell expressing the polypeptide of the invention and having a property or function ascribable to the polypeptide;
contacting the cell with a composition comprising a candidate substance; and deterniining whether the substance alters the property or function ascribable to the polypeptide; whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition devoid of the substance, the substance is identified as a potential therapeutic agent.
In another embodiment, the invention involves a method for screening for a modulator of activity or of latency or predisposition to a pathology associated with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
N0:2n, wherein n is an integer between 1 and 141, the method including administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide of the invention, wherein the test animal recornbinantly expresses the polypeptide of the invention; measuring the activity of the polypeptide in the test animal after administering the 1 S test compound; and comparing the activity of the protein in the test animal with the activity of the polypeptide in a control animal not administered the polypeptide, wherein a change in the activity of the polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of, or predisposition to, a pathology associated with the polypeptide of the invention. The recombinant test animal could express a test protein transgene or express the transgene under the control of a promoter at an increased level relative to a wild-type test animal The promoter may or may not b the native gene pxomoter of the transgene.
In another embodiment, the invention involves a method fox modulating the activity of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID
2S N0:2n, wherein n is an integer between I and 141, the method including introducing a cell sample expressing the polypeptide with a compound that binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide.
In another embodiment, the invention involves a method of treating or preventing a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integex between 1 and 141, the method including administering the polypeptide to a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject. The subject could be human.

In another embodiment, the invention involves a method of treating a pathological state in a mammal, the method including 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 having the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141 or a biologically active fragment thereof.
In another embodiment, the invention involves an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 141; a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141 wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141; a variant of the amino acid sequence selected from the group consisting of SEQ
ID N0:2n, wherein n is an integer between 1 and 141, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15%
of the amino acid residues in the sequence are so changed; a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141 or any variant of the polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and the complement of any of the nucleic acid molecules.
In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 141, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.
In another embodiment, the invention involves an isolated nucleic acid molecule including a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 141 that encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.
In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence S selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 141, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2n-1, wherein n is an integer between 1 and 141.
In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein ri is an integer between 1 and 141, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1, wherein n is an integer 1 S between 1 and 141; a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; a nucleic acid fragment of the sequence selected from the group consisting of SEQ
ID N0:2n-l, wherein n is an integer between 1 and 141; and a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 1 S% of the nucleotides are so changed.
In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 141, wherein the nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141, or a complement of the nucleotide sequence.
In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a matuxe foam of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 141, wherein the nucleic acid molecule has a nucleotide sequence in which any nucleotide specified in the coding sequence of the chosen nucleotide sequence is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides in the chosen coding sequence are so changed, an isolated second polynucleotide that is a complement of the first polynucleotide, or a fragment of any of them.
In another embodiment, the invention includes a vector involving the nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 arid 141. This vectox can have a promoter operably linked to the nucleic acid molecule. This vector can be located within a cell.
In another embodiment, the invention involves a method for determining the presence 1 S or amount of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID N0:2n, wherein n is an integer between 1 and I41 in a sample, the method including providing the sample; introducing the sample to a probe that binds to the nucleic acid molecule; and determining the presence or amount of the probe bound to the nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in the sample. The presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type. The cell type can be cancerous.
In another embodiment, the invention involves a method for determining the presence of or predisposition for a disease associated with altered levels of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 141 in a first mammalian subject, the method including measuring the amount of the nucleic acid in a sample from the first mammalian subject; and comparing the amount of the nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

The invention further provides an antibody that binds immunospecifically to a NOVX
polypeptide. The NOVX antibody may be monoclonal, humanized, or a fully human antibody. Preferably, the antibody has a dissociation constant for the binding of the NOVX
polypeptide to the antibody less than 1 x 10-9 M. More preferably, the NOVX
antibody neutralizes the activity of the NOVX polypeptide.
In a further aspect, the invention provides for the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, associated with a NOVX polypeptide. Preferably the therapeutic is a NOVX
antibody.
Tn 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, arid other related compounds. The sequences are collectively referred to herein as "NOVX nucleic acids" or "NOVX polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NOVX nucleic acids and their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers SEQ ID SEQ ID

NOVX Internal NO NO Homology AssignmentIdentification(nucleic (amino acid acid Kunitz-type Protease Inhibitor 2 1 a CG103134-O11 2 recursor-like Kunitz-type Protease Inhibitor 2 1b CG103134-023 4 recursor-like 2a CG103322-O1S 6 CD82 Anti en-like 2b CG103322-027 8 CD82 Anti en-like Multi-pass Membrane Protein-3a CG151575-Ol9 10 like Multi-pass Membrane Protein-3b CGISI575-0211 12 lik e 4a CGI51608-Ol13 14 T a 1b Membrane Protein-like 4b CGI51608-0215 I6 T a 1b Membrane Protein-like Sa CG152323-OI17 18 Laminin beta 4-like Sushi Domain-containing 6a CG153011-O119 20 Membrane Protein-like 7a CGI53042-Ol21 22 RIK Protein-like 7b CG153042-0223 24 RIK Protein-like 8a CG153179-Ol25 26 Membrane Protein-like 9a CG153403-O127 2g Dickkopf Related Protein-4 Precursor-like 9b CG153403-0229 30 Dickkopf Related Protein-4 Precursor-like Dickkopf Related Protein-4 9c 30503755831 32 Precursor-like 9d 30503751233 34 Dickkopf Related Protein-4.

Precursor-like 10a CG153424-O13S 36 IGFBP4-like l la CG157567-Ol37 3g Leucine Rich Repeat Protein-like 12a CG157760-O139 40 Placental S ecific Protein 1-like 12b CG157760-0241 42 Placental S ecific Protein 1-like 13a CG157844-O143 44 Type ~ Membrane Protein-like 14a CG1581I4-Ol45 46 Silver-like 15a CG158553-O147 48 E o oietin Rece tor-like 15b CG158553-Ol49 50 E o oietin Rece tor-like 15c CGI58553-OZS 1 52 E o oietin Rece tor-like 15d CG158553-0353 54 E o oietin Rece tor-like 16a CG158983-Ol55 56 Chloride Channel-like 16b CG158983-0257 58 Chloride Channel-like 16c CG158983-0359 60 Chloride Channel-like 16d CG158983-O161 62 Chloride Channel-like 16e CG158983-O163 64 Chloride Channel-like 17a CG159015-O165 66 Secreted Protein-like 17b CG159015-0267 68 Secreted Protein-like 17c CG159015-0369 70 Secreted Protein-like 17d CG159015-0471 72 Secreted Protein-like 18a CG173007-Ol73 74 Prolactin Receptor Precursor-like 19a CG173357-O175 76 ~'~oglobulin Domain Containin Protein-like 20a CG50387-0177 78 Connexin 46 20b CG50387-0379 80 Connexin 46 20c CG50387-0281 82 Connexin 46 21a CG52113-Ol83 84 Notch4-like 21b CG52113-0685 8b Notch4-like 21c 274054261 87 88 Notch4-like 21d 274054299 89 90 Notch4-like 21e 274054261 91 92 Notch4-like 21f 274054299 93 94 Notch4-like 21 CG52113-029S 96 Notch4-like 21h CG52113-0397 98 Notch4-like 21i CGS2113-0499 100 Notch4-like 21' CG52113-OS101 I02 Notch4-like 22a CGS7542-01103 104 Cadherin-23 Precursor-like 22b 169258612 105 106 Cadherin-23 Precursor-like 22c 169258615 107 108 Cadherin-23 Precursor-like 22d 169258621 109 110 Cadherin-23 Precursor-like 22e 174307774 111 112 Cadherin-23 Precursor-like 23a CGS7774-O1113 114 TRNFR-19 Protein 23b 167200132 115 116 TRNFR-19 Protein 23c 167200144 117 118 TRNFR-19 Protein 23d 169252408 119 120 TRNFR-19 Protein 23e 169252412 121 122 TRNFR-19 Protein 23f 169252424 I23 124 TRNFR-19 Protein 23 169252469 125 126 TRNFR-19 Protein 23h 169252475 127 128 TRNFR-19 Protein 23i 169252481 129 130 TRNFR-19 Protein 23' 169252485 131 132 TRNFR-19 Protein 23k 169252492 133 134 TRNFR-19 Protein 2_31 174104491 I35 136 TRNFR-19 Protein _ 169252509 137 138 TRNFR-19 Protein 23m 23n 16 139 140 TRNFR-19 Protein 230 _ 14 142 TRNFR-19 Protein _ 1 _ _ 23 169252524 143 144 TRNFR-19 Protein 23 169252528 I45 146 TRNFR-19 Protein 23r 169252547 147 148 TRNFR-19 Protein 23s 169252557 149 150 TRNFR-19 Protein 23t 174104491 151 152 TRNFR-19 Protein 23u CGS7774-02153 154 TRNFR-19 Protein 23v CGS7774-0315S 156 TRNFR-19 Protein 23w CGS7774-04157 158 TRNFR-19 Protein 23x CGS7774-OS159 160 TRNFR-19 Protein 23 CGS7774-06161 162 TRNFR-19 Protein 23z CG57774-07163 164 TRNFR-19 Protein 23aa CG57774-08165 166 TRNFR-19 Protein 23ab CG57774-09167 168 TRNFR-19 Protein 23ac CG57774-10169 170 TRNFR-19 Protein 23ad CG57774-11171 172 TRNFR-19 Protein 23ae CG57774-12173 174 TRNFR-19 Protein 23af CGS7774-13175 176 TRNFR-19 Protein 24a CG89285-Ol177 178 AI ha-1-Antich otr sin-like 24b CG8928S-04179 180 Al ha-1-.Antich ao sin-like 24c CG8928S-03181 182 A1 ha-1-Antich o sin-Iike 24d 306418132 183 184 AI ha-1-Antich o sin-like 24e CG89285-02185 186 A1 ha-1-Antich o sin-like 25a CG57094-OI187 188 Human an 'o oietin-like 25b 170075926 189 190 Human an 'o oietin-like 25c 164225601 191 192 Human an 'o oietin-like 25d 164225637 193 194 Human an 'o oietin-like 25e 170075926 195 196 Human an 'o oietin-like 25f 254120574 197 198 Human an 'o oietin-like 2S 254156650 199 200 Human an 'o oietin-like 25h 254500366 201 202 Human an 'o oietin-like 25i 226679956 203 204 Human an 'o oietin-like 2S' 254500319 205 206 Human an 'o oietin-like 25k .254500445207 208 Human an 'o oietin-Like 25l 248210290 209 210 Hurnan an 'o oietin-like 25m 252514148 211 212 Human an 'o oietin-like 25n 252514189 213 214 Human an 'o oietin-like 250 252514198 215 216 Human an 'o oietin-like 25 252514202 217 218 Human an 'o oietin-like 2S 228039766 219 220 Human an 'o oietin-like 25r 226679952 221 222 Human an 'o oietin-like 25s CGS7094-02223 224 Human an 'o oietin-like 25t CGS7094-03225 226 Human an 'o oietin-like 25u CGS7094-04227 228 Human an 'o oietin-like 25v CG57094-OS229 230 Human an 'o oietin-like 25w CGS7094-06231 232 Human an 'o oietin-like 25x CGS7094-07233 234 Human an 'o oietin-like 2S CGS7094-08235 236 Human an 'o oietin-like 25z CG57094-09237 238 Human an 'o oietin-like 2Saa CGS7094-10239 240 Human an 'o oietin-like 2Sab CGS7094-11241 242 Human an 'o oietin-like 2Sac CGS7094-12243 244 Human an 'o oietin-like 2Sad CGS7094-13245 246 Human an 'o oietin-like 26a CGS 1523-OS247 248 Endozepine Related Protein Precursor-like 26b CGS 1523- 249 250 Endozepine Related OS 164786042 Protein Precursor-like 26c CGS 1523- 2S I 252 Endozepine Related OS 164732479 Protein Precursor-like 26d CGS 1523- 253 254 Endozepine Related OS 164732506 Protein Precursor-like 26e CGS1S23- 25S 256 EndozepineRelatedProtein OS 164732693 Precursor-like 26f CG51523- 257 258 Endozepine Related OS 164732709 Protein Precursor-like 26g CG51523- 259 260 Endozepine Related OS 164718189 Protein Precursor-Like 26h CGS1S23- 261 262 EndozepineRelatedProtein OS 164718193 Precursor-like 26i CGS 1523- 263 264 Endoze ine Related Protein OS 164718197 Precursor-like 26j CGS 1523- 265 266 Endozepine Related Protein OS 164718205 Precursor-like 26k CG51523- 267 268 Endozepine Related Protein OS 164718209 Precursor-like 261 CG51523- 269 270 Endozepine Related Protein OS 164718213 Precursor-like 26m CGS 1523- 271 272 Endozepine Related Protein OS 166190452 Precursor-like 26n CG51523- 273 274 Endozepine Related Protein OS 166190467 Precursor-like 26o CG51523- 275 276 Endozepine Related Protein OS 166190475 Precursor-like 26p CG51523- 277 278 Endozepine Related Protein OS 166190498 Precursor-like 26q CGSI523- 279 280 Endozepine Related Protein OS 166190460 Precursor-like 26r CG51523- 281 282 Endozepine Related Protein OS 166190483 Precursor-like Table A indicates the homology of NOVX polypeptides to known protein families.
Thus, the nucleic acids and polypeptides, antibodies and related compounds according to the invention corresponding to a NOVX as identified in column 1 of Table A will be useful in therapeutic and diagnostic applications implicated in, for example, pathologies and disorders associated with the known protein families identified in column S of Table A.
Pathologies, diseases, disorders and condition and the like that are associated with NOVX sequences include, but are not limited to: ~e,~~, r ~art~~c~~mypp~t~y, a~rpsc~eraszs rY n a', v~h''~' c l~' A~ ~cariald f a ~d etits arte 's i~~n.~~. stexio s sub ~~~ .
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ndi ' ~- , ell t1 st ids order' a.-s Ia ~ h chro c'~chseas ~s and~~ aiao 'y~c ~tcexs :a~ w. ~~
~a. o Wa, ln~.: . 5.. ... .5 S ,.0G , ~et~'W ~ , .:. . ,. ~.... . _: _..~~ . ._ _.
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:.0:~:
. "._ a ~ '~ t ~. -, d n S'l'iCb~ ~S tx'allS laxJtatiO~l 'ilell~'O IOteG~~t~n ~~r~i.ll , -Clx.'~'e Y
BIl rat'~pn ~2 VZ l ~ ~~.1? ..
_:: ... _ __~_.._._.. __ ._ ~. . _, :.~ .. ....:. _._ . ~ :.._ . ..~'a ,..._..m~._ __,.;.._.. ~. ,.. _.:......_ .....z :..,~.~;
NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX
nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
Consistent with other known members of the family of proteins, identified in column 5 of Table A, the NOVX polypeptides of the present invention show homology to, and contain domains that are characteristic of, other members of such protein families. Details of the sequence relatedness and domain analysis for each NOVX are presented in Example A.
The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit diseases associated with the protein families listed in Table A.
The NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details of the expression analysis for each NOVX are presented in Example C.
Accordingly, the NOVX nucleic acids, polypeptides, antibodies and related compounds according to the invention will have diagnostic and therapeutic applications in the detection of a variety of diseases with differential expression in normal vs. diseased tissues, e.g.
detection of a variety of cancers.
Additional utilities for NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
NOVX clones NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX
nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.

The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e.g., by protein or gene therapy.
Pathological conditions can be diagnosed by determining the amount of the new protein in a sample or by determining the presence of mutations in the new genes. Specific uses are described for each of the NOVX genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.
°The NOVX nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (t) 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 141; (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 141, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) an amino acid sequence selected from the group consisting of SEQ
ID NO: 2n, wherein n is an integer between 1 and 141; (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; and (e) a fragment of any of (a) through (d).
In another specific embodiment, the invention includes an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of the amino acid sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 141; (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 141 wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 141; (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 141, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; (e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ Il? NO: 2n, wherein n is an integer between 1 and 141 or any variant of said polypeptide wherein any amino acid of the chosen sequence is changed to.a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and (f) the complement of any of said nucleic acid molecules.
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-I, wherein n is an integer between 1 and I41; (b) a nucleotide sequence 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 141 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; (c) a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 141; 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 141 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.
N~VX 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 axe nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as FCR
primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA
or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.
A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or pxoprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell (e.g., host cell) in which the 1 S 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 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature"
form of a polypeptide or pxotein may arise from a step of post-translational modification other than a 2S proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probe", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), about 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences.
Longer length pxobes are generally obtained from a natural or recombinant source, are highly specific, and l~

much slower to hybridize than shorter-length oligomer probes. Probes may be single-stranded or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as used herein, is a nucleic acid that is separated from other nucleic acid molecules which are present in the natural source of tha 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 genornic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain Less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated"
nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, or of chemical precursors ox other chemicals.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:2h-I, wherein h is an integer between 1 and 141, or a complement of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID N0:2yi-1, wherein n is an integer between I
and 141, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning technnques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR
CLONING: A
LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 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 with appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID N0:2ra-1, wherein n is an integer between 1 and 141, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID
N0:2rr-1, wherein h is an integer between 1 and 141, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141, that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID N0:2ra-1, wherein n is an integer between 1 and 141, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
A "fragment" provided herein is defined as a sequence of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, and is at most some portion less than a full length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid ox amino acid sequence of choice.
A full-length NOVX clone is identified as containing an ATG translation start codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence lacking an ATG

start codon therefore encodes a truncated C-terminal fragment of the respective NOVX
polypeptide, and requires that the corresponding full-length cDNA extend in the 5' direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective NOVX
polypeptide, and requires that the corresponding full-length cDNA extend in the 3' direction of the disclosed sequence.
A "derivative" is a nucleic acid sequence or amino acid sequence formed from the native compounds either directly, by modification or partial substitution. An "analog" is a nucleic acid sequence or amino acid sequence that has a stxucture similar to, but not identical to, the native compound, e.g. they differs from it in respect to certain components or side chains. Analogs may be synthetic or derived from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. A
"homolog" is a nucleic acid sequence or amino acid sequence of a particular gene that is derived from different species.
1 S Derivatives and analogs may be full length or other than full length.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95%
identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CU~?NT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX
polypeptide of species other than humans, including, but not limited to:
vertebrates, and thus can include, e.g., frog, mouse, xat, 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 N0:2ra-1, wherein n is an integer between 1 and 141, as well as a polypeptide possessing NOVX
biological activity. Various biological activities of the NOVX proteins are described below.
A NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one of the three "stop" colons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF 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 often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/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 N0:2rz-1, wherein h is an integer between 1 and 141; or an anti-sense strand nucleotide sequence of SEQ ID
N0:2n-1, wherein ra is an integer between 1 and 141; or of a naturally occurring mutant of SEQ ID N0:2h-1, wherein h is an integer between 1 and 141.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe has a detectable label attached, e.g. the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which rnis-express a NOVX
protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX
gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of a NOVX polypeptide"
refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion of SEQ ID
N0:2n-1, wherein n is an integer between 1 and 141, that encodes a polypeptide having a NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID N0:2n-l, wherein n is an integer between 1 and 141, due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID NO:2ya-l, wherein n is an integer between 1 and 141. 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 1D N0:2n, wherein n is an integer between 1 and 141.
In addition to the human NOVX nucleotide sequences of SEQ ID NO:2n-l, wherein n is an integer between 1 and 141, it will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX
polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX
protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations.and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX
polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from a human SEQ ID N0:2n-1, wherein ra is an integer between 1 and 141, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX
cDNAs of the invention can be isolated based on their homology to the human NOVX
nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2ra-1, wherein ya is an integer between 1 and 141. 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 I 5 about 65% homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Trn, 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 BIOLOGY, 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, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO:2h-1, wherein n is an integer between 1 and 141, 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 N0:2h-1, wherein h is an integer between 1 and 141, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X 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 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 N0:2ra-l, wherein n is an integer between 1 and 141, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02%
PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH
7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used axe well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Pf-oc Natl Acad Scz 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 sequences of SEQ ID N0:2fz-1, wherein ra is an integer between 1 and 141, thereby leading to changes in the amino acid sequences of the encoded NOVX
protein, without altering the functional ability of that NOVX protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID N0:2T2, wherein h is an integex between 1 and 141. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding NOVX
proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID N0:2ya-1, wherein fa is an integer between 1 and 141, yet retain biologieal~activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 40% homologous to the amino acid sequences of SEQ ID N0:2n, wherein n is an integer between 1 and 141.
Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID
N0:2~, wherein n is an integer between 1 and 141; more preferably at least about 70%
homologous to SEQ ID N0:2ya, wherein r~ is an integer between 1 and 141; still more preferably at least about 80% homologous to SEQ ID N0:2n, wherein h is an integer between 1 and 141; even more preferably at least about 90% homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 141; and most preferably at least about 95%
homologous to SEQ ID N0:2ra, wherein fa is an integer between 1 and 141.

An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID N0:2n, wherein n is an integer between 1 and 141, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID N0:2ra-1, wherein ra is an integer between 1 and 141, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced any one of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVA coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX
biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID NO:2n-1, wherein n is an integer between 1 and 141, 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).
Interfering RNA
In one aspect of the invention, NOVX gene expression can be attenuated by RNA
interference. One approach well-known in the art is short interfering RNA
(siRNA) mediated gene silencing where expression products of a NOVX gene are targeted by specific double stranded NOVX derived siRNA nucleotide sequences that are complementary to at least a 19-25 nt long segment of the NOVX gene transcript, including the 5' untranslated (UT) region, the ORF, or the 3' UT region. See, e.g., PCT applications WO00/44895, W099132619, WO01/75164, WO01/92513, WO 01/29058, WO01/89304, W002/I6620, and W002/29858, each incorporated by reference herein in their entirety. Targeted genes can be a NOVX gene, or an upstream or downstream modulator of the NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX gene include, e.g., a transcription factor that binds the NOVX gene promoter, a kinase or phosphatase that interacts with a NOVX
polypeptide, and polypeptides involved in a NOVX regulatory pathway.
According to the methods of the present invention, NOVX gene expression is silenced using short interfering RNA. A NOVX polynucleotide according to the invention includes a siRNA polynucleotide. Such a NOVX siRNA can be obtained using a NOVX
polynucleotide sequence, for example, by processing the NOVX xibopolynucleotide sequence in a cell-free system, such as but not limited to a Drosophila extract, or by transcription of recombinant double stranded NOVX RNA ox by chemical synthesis of nucleotide sequences homologous to a NOVX sequence. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorpoxated herein by reference in its entirety. When synthesized, a typical 0.2 micromolar-scale RNA synthesis provides about 1 milligram of siRNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.

The most efficient silencing is generally observed with siRNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to have a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang makes an additional small contribution to the specificity of siRNA target recognition. The contribution to specificity is localized to the unpaired nucleotide adjacent to the first paired bases. In one embodiment, the nucleotides in the 3' overhang are ribonucleotides. In an alternative embodiment, the nucleotides in the 3' overhang are deoxyribonucleotides. Using 2'-deoxyribonucleotides in the 3' overhangs is as efficient as using ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely more nuclease resistant.
A contemplated recombinant expression vector of the invention comprises a NOVX
DNA molecule cloned into an expression vector comprising operatively-linked regulatory sequences flanking the NOVX sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands. An RNA molecule that is antisense to NOVX mRNA is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the NOVX mRNA is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA). The sense and antisense strands may hybridize in vivo to generate siRNA constructs for silencing of the NOVX gene. Alternatively, two constructs can be utilized to create the sense and anti-dense strands of a siRNA construct. Finally, cloned DNA can encode a construct having secondary structure, wherein a single transcript has both the sense and complementary antisense sequences from the target gene or genes. In an example of this embodiment, a hairpin RNAi product is homologous to all or a portion of the target gene. In another example, a hairpin RNAi product is a siRNA. The regulatory sequences flanking the NOVX sequence may be identical or may be different, such that their expression may be modulated independently, or in a temporal or spatial manner.
In a specific embodiment, siRNAs are transcribed intracellularly by cloning the NOVX gene templates into a vector containing, e.g., a RNA pol III
transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA Hl. One example of a vector system is the GeneSuppressorTM RNA Interference kit (commercially available from Imgenex). The U6 and H1 promoters are members of the type III class of Pol III
promoters.
The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for Hl promoters is adenosine. The termination signal for these promoters is defined by five consecutive thyrnidines. The transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed siRNA, which is similar to the 3' overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript.
A siRNA vector appears to have an advantage over synthetic siRNAs where long term knock-down of expression is desired. Cells transfected with a siRNA expression vector would experience steady, long-term mRNA inhibition. In contrast, cells transfected with exogenous synthetic siRNAs typically recover from mRNA suppression within seven days or ten rounds of cell division. The long-term gene silencing ability of siRNA
expression vectors may provide for applications in gene therapy.
In general, siRNAs are chopped from longer dsRNA by an ATP-dependent ribonuclease called DICER. DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular proteins into an endonuclease complex. In vitro studies in Drosophila suggest that the siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex, called an RNA-induced silencing complex (RISC), which contains an endoribonuclease that is distinct from DICER.
RISC uses the sequence encoded by the antisense siRNA strand to find and destroy mRNAs of complementary sequence. The siRNA thus acts as a guide, restricting the ribonuclease to cleave only mRNAs complementary to one of the two siRNA strands.
A NOVX mRNA region to be targeted by siRNA is generally selected from a desired NOVX sequence beginning 50 to100 nt downstream of the start codon.
Alternatively, 5' or 3' UTRs and regions nearby the start codon can be used but are generally avoided, as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC
endonuclease complex. An initial BLAST homology search for the selected siRNA sequence is done against an available nucleotide sequence library to ensure that only one gene is targeted.
Specificity of taxget recognition by siRNA duplexes indicate that a single point mutation located in the paired region of an siRNA duplex is sufficient to abolish target mRNA
degradation. See, Elbashir et al. 2001 EMBO J. 20(23):6877-88. Hence, consideration should be taken to accommodate SNPs, polymorphisms, allelic variants or species-specific variations when targeting a desired gene.
In one embodiment, a complete NOVX siRNA experiment includes the proper negative control. A negative control siRNA generally has the same nucleotide composition as the NOVX siRNA but lack significant sequence homology to the genome.
Typically, one would scramble the nucleotide sequence of the NOVX siRNA and do a homology search to make sure it lacks homology to any other gene.
Two independent NOVX siRNA duplexes can be used to knock-down a target NOVX
gene. This helps to control for specificity of the silencing effect. In addition, expression of two independent genes can be simultaneously knocked down by using equal concentrations of different NOVX siRNA duplexes, e.g., a NOVX siRNA and an siRNA for a regulator of a NOVX gene or polypeptide. Availability of siRNA-associating proteins is believed to be more limiting than target mRNA accessibility.
A targeted NOVX region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (N19) residues (e.g., AA(N19)TT).
A desirable spacer region has a G/C-content of approximately 30% to 70%, and more preferably of about 50%. If the sequence AA(N19)TT is not present in the target sequence, an alternative target region would be AA(N21 ). The sequence of the NOVX sense siRNA
corresponds to (N19)TT or N21, respectively. In the latter case, conversion of the 3' end of the sense siRNA to TT can be performed if such a sequence does not naturally occur in the NOVA polynucleotide. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs.
Symmetric 3' overhangs may help to ensure that the siRIVPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs. See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200, incorporated by reference herein in its entirely. The modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA
strand guides target recognition.
Alternatively, if the NOVX target mRNA does not contain a suitable AA(N21) sequence, one may search for the sequence NA(N21). Further, the sequence of the sense strand and antisense strand may still be synthesized as 5' (Nl9)TT, as it is believed that the sequence of the 3'-most nucleotide of the antisense siRNA does not contribute to specificity.
Unlike antisense or ribozyme technology, the secondary structure of the target mRNA does not appear to have a strong effect on silencing. See, Harborth, et al. (2001) J. Cell Science 114: 4557-4565, incorporated by reference in its entirety.
Transfection of NOVX siRNA duplexes can be achieved using standard nucleic acid transfection methods, for example, OLIGOFECTAMINE Reagent (commercially available from Invitrogen). An assay for NOVX gene silencing is generally performed approximately 2 days after transfection. No NOVX gene silencing has been observed in the absence of transfection reagent, allowing for a comparative analysis of the wild-type and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-well plate, approximately 0.84 wg of the siRNA duplex is generally sufficient. Cells are typically seeded the previous day, and are transfected at about 50% confluence. The choice of cell culture media and conditions are routine to those of skill in the art, and will vary with the choice of cell type.
The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful NOVX silencing. The efficiency of transfection needs to be carefully examined for each new cell line to be used. Preferred cell are derived from a mammal, more preferably from a rodent such as a rat or mouse, and most preferably from a human. Where used for therapeutic treatment, the cells are preferentially autologous, although non-autologous cell sources are also contemplated as within the scope of the present invention.
For a control experiment, transfection of 0.84 ~.g single-stranded sense NOVX
siRNA
will have no effect on NOVX silencing, and 0.84 wg antisense siRNA has a weak silencing effect when compared to 0.84 ~,g of duplex siRNAs. Control experiments again allow for a comparative analysis of the wild-type and silenced NOVX phenotypes. To control for transfection efficiency, targeting of common proteins is typically performed, for example targeting of lamin A/C or transfection of a CMV-driven EGFP-expression plasmid (e.g.
commercially available from Clontech). In the above example, a determination of the fraction of larnin A/C knockdown in cells is determined the next day by such techniques as immunofluorescence, Western blot, Northern blot or other similar assays for protein expression or gene expression. Lamin A/C monoclonal antibodies may be obtained from Santa Cruz Biotechnology.
Depending on the abundance and the half life (or turnover) of the targeted NOVX
polynucleotide in a cell, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no NOVX knock-down phenotype is observed, depletion of the NOVX polynucleotide may be observed by immunofluorescence or Western blotting.
If the NOVX polynucleotide is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. If no knock-down of the targeted protein is observed, it may be desirable to analyze whether the target mRNA (NOVX or a NOVX
upstream or downstream gene) was effectively destroyed by the transfected siRNA duplex.
Two days after transfection, total RNA is prepared, reverse transcribed using a target-specific primer, and PCR-amplified with a primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA yet undetectable reduction of target protein may indicate that a large reservoir of stable NOVX protein may exist in the cell.
Multiple transfection in sufficiently long intervals may be necessary until the target protein is finally depleted to a point where a phenotype may become apparent. If multiple transfection steps are required, cells are split 2 to 3 days after transfection. The cells may be transfected immediately after splitting.
An inventive therapeutic method of the invention contemplates administering a NOVX siRNA construct as therapy to compensate for increased or aberrant NOVX
expression or activity. The NOVX ribopolynucleotide is obtained and processed into siRNA
fragments, or a NOVX siRNA is synthesized, as described above. The NOVX
siRNA'is administered to cells or tissues using known nucleic acid transfection techniques, as described above. A NOVX siRNA specific for a NOVX gene will decrease or knockdown NOVX transcription products, which will lead to reduced NOVX polypeptide production, resulting in reduced NOVX polypeptide activity in the cells or tissues.
The present invention also encompasses a method of treating a disease or condition associated with the presence of a NOVX protein in an individual comprising administering to the individual an RNAi construct that taxgets the mRNA of the protein (the mRNA that encodes the protein) for degradation. A specific RNAi construct includes a siRNA or a double stranded gene transcript that is processed into siRNAs. Upon treatment, the target protein is not produced or is not produced to the extent it would be in the absence of the treatment.
Where the NOVX gene function is not correlated with a known phenotype, a control sample of cells or tissues from healthy individuals provides a reference standard for determining NOVX expression levels. Expression levels are detected using the assays described, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the like. A subject sample of cells or tissues is taken from a mammal, preferably a human subject, suffering from a disease state. The NOVX ribopolynucleotide is used to produce siRNA
constructs, that are specific for the NOVV~ gene product. These cells or tissues are treated by administering NOVX siRNA's to the cells or tissues by methods described for the transfection of nucleic acids into a cell or tissue, and a change in NOVX
polypeptide or polynucleotide expression is observed in the subject sample relative to the control sample, using the assays described. This NOVX gene knockdown approach provides a rapid method for determination of a NOVX minus (NOVX-) phenotype in the treated subject sample. The NOVX' phenotype observed in the treated subject sample thus serves as a marker for monitoring the course of a disease state during treatment.
In specific embodiments, a NOVX siRNA is used in therapy. Methods for the generation and use of a NOVX siRNA are known to those skilled in the art.
Example techniques are provided below.
Production of RNAs Sense RNA (ssRNA) and antisense RNA (asRNA) of NOVX are produced using known methods such as transcription in RNA expression vectors. In the initial experiments, the sense and antisense RNA are about 500 bases in length each. The produced ssRNA and asRNA (0.5 p,M) in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCI were heated to 95° C for 1 min then cooled and annealed at room temperature for 12 to 16 h. The RNAs are precipitated and resuspended in lysis buffer (below). To monitor annealing, RNAs are electrophoresed in a 2% agarose gel in TBE buffer and stained with ethidium bromide. See, e.g., Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
(1989).
Lysate Preparation Untreated rabbit reticulocyte lysate (Ambion) are assembled according to the manufacturer's directions. dsRNA is incubated in the lysate at 30° C
for 10 min prior to the addition of mRNAs. Then NOVX mRNAs are added and the incubation continued for an additional 60 min. The molar ratio of double stranded RNA and mRNA is about 200:1. The NOVX mRNA is radiolabeled (using known techniques) and its stability is monitored by gel electrophoresis.
In a parallel experiment made with the same conditions, the double stranded RNA is internally radiolabeled with a 32P-ATP. Reactions are stopped by the addition of 2 X
proteinase K buffer and deproteinized as described previously (Tuschl et al., Genes Dev., 13:3191-3197 (1999)). Products are analyzed by electrophoresis in 15% or 18%
polyacrylamide sequencing gels using appropriate RNA standards. By monitoring the gels for radioactivity, the natural production of 10 to 25 nt RNAs from the double stranded RNA
can be determined.
The band of double stranded RNA, about 21-23 bps, is eluded. The efficacy of these 21-23 mers for suppressing NOVX transcription is assayed in vitro using the same rabbit reticulocyte assay described above using SO nanomolar of double stranded 21-23 mer for each assay. The sequence of these 21-23 mers is then determined using standard nucleic acid sequencing techniques.
RNA Preparation 21 nt RNAs, based on the sequence determined above, are chemically synthesized using Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo, Germany).
Synthetic oligonucleotides are deprotected and gel-purified (Elbashir, Lendeckel, & Tuschl, Genes 8e Dev. 15, 188-200 (2001)), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA) purification (Tuschl, et al., Biochemistry, 32:11658-11668 (1993)).
These RNAs (20 p.M) single strands are incubated in annealing buffer (100 mM
potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C followed by 1 h at 37° C.
Cell Culture A cell culture known in the art to regularly express NOVX is propagated using standard conditions. 24 hours before transfection, at approx. 80% confluency, the cells are trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3 X 105 cells/ml) and transferred to 24-well plates (500 ml/well). Transfection is performed using a commercially available lipofection kit and NOVX expression is monitored using standard techniques with positive and negative control. A positive control is cells that naturally express NOVX while a negative control is cells that do not express NOVX. Base-paired 21 and 22 nt siRNAs with overhanging 3' ends mediate efficient sequence-specific mRNA degradation in lysates and in cell culture. Different concentrations of siRNAs are used. An efficient concentration for suppression in vitro in mammalian culture is between 25 nM to 100 nM final concentration.
This indicates that siRNAs are effective at concentrations that are several orders of rriagnitude below the concentrations applied in conventional antisense or ribozyme gene targeting experiments.
The above method provides a way both for the deduction of NOVX siRNA sequence and the use of such siRNA for in vitro suppression. In vivo suppression may be performed using the same siRNA using well known in vivo transfection or gene therapy transfection techniques.
Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2n-1, wherein ra is an integer between 1 and 141, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or S00 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 rz is an integer between 1 and 141, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID N0:2fz-1, wherein r~
is an integer between 1 and 141, 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 (r. 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 mltNA.
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-occurnng nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluxacil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a ~25 subject or generated i~a 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 delivered to cells using the vectors described herein.
To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987.
Nucl. Acids Res. 15:
6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., moue, et al. 1987. Nucl. 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 carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX
mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX cDNA disclosed herein (i.e., SEQ ID N0:2ra-1, wherein n is an integer between 1 and 141). 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 region of the NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84;
Helene, et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioorg Med Chem 4: S-23. As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleotide bases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomer can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93:
14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
PNAs of NOVA 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., 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra;
Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of dxug 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 intexact 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 bases, and orientation (see, Hyrup, et al., 1996.

supra). The synthesis of PNA-DNA chimeras can be performed as described in I3yrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., S'-(4-methoxytrityl)amino-S'-deoxy-thymidine phosphoramidite, can be used between the PNA arid 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. Lett. 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 (see, e.g., I~rol, et al., 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
N0:2ra, wherein n is an integer between 1 and 141. The invention also includes a mutant or 2S variant protein any of whose residues may be changed from the corresponding residues shown in any one of SEQ ID N0:2n, wherein n is an integer between 1 and 141, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
In general, a NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof.
Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX
antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
Tn another embodiment, NOVX proteins are produced by recombinant DNA
techniques.
Alternative to recombinant expression, a NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparations of NOVX proteins having less than about 30%
(by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10%of non-NOVX proteins, and most preferably less than about 5% of non-NOVX
proteins.
When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence of SEQ ID N0:2n, wherein n is an integer between 1 and 141) that include fewer amino acids than the full-length NOVX
proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A
biologically-active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID
N0:2ra, wherein n is an integer between 1 and 141. In other embodiments, the NOVX
protein is substantially homologous to SEQ ID N0:2ra, wherein n is an integer between 1 and 141, and retains the functional activity of the protein of SEQ ID N0:2n, wherein n is an integer between 1 and 141, 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 N0:2ra, wherein n is an integer between 1 and 141, and retains the functional activity of the NOVX proteins of SEQ ID N0:2n, wherein n is an integer between 1 and 141.
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 identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMoI Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence of SEQ ID N0:2h-l, wherein h is an integer between 1 and.141.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins The invention also provides NOVX chimeric or fusion proteins. As used herein, a NOVX "chimeric protein" or "fusion protein" comprises a NOVA 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
N0:2h, wherein h is an integer between 1 and 141, 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 two biologically-active portions of a NOVX protein. In yet another embodiment, a NOVX fusion protein comprises at least three biologically-active portions of a NOVX
protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX
polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX
polypeptides.
In another embodiment, the fusion protein is a NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased thxough 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 irnmunoglobulin 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 NOVA ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction ih vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX
cognate ligand. Inhibition of the NOVX,ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand.
A NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chirneric 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 occurring form of the NOVX proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i. e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then Iigated 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. Annu. Rev. Biochenz. 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 S1 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 (RENT), 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. Aced. Sci. USA 89:
7811-7815;
Delgrave, et al., 1993. Protein EhgineeYitag 6:327-331.

Anti-NOVX Antibodies Included in the invention are antibodies to NOVX proteins, or fragments of NOVX
proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab~ and F~ab~>z fragments, and an Fab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
Certain classes have subclasses as well, such as IgGI, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence of SEQ ID
N0:2n, wherein h is an integer between 1 and 141, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human NOVX protein sequence will indicate which regions of a NOVX polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
3824-3828;
Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
The term "epitope" includes any protein determinant capable of specific binding to an imrnunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. A NOVX polypeptide or a fragment thereof comprises at least one antigenic epitope. An anti-NOVX antibody of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (KD) is <_ 1 pM, preferably <_ 100 nM, more preferably <_ 10 nM, and most preferably <_ 100 pM to about 1 pM, as 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 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 immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar imrnunostimulatory 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 immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia.
Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked irnmunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
It is an objective, especially important in therapeutic 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 murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA
also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (LT.S. Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')z 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 irnmunoglobulin. 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; Riechrnann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Human Antibodies Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridorna 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 al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
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, 845-51 (1996));
Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse~ as disclosed in PCT publications WO

and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent 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 99153049.
Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g.; Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~~b72 fragment produced by pepsin digestion of an antibody molecule;
(ii) an Fab fragment generated by reducing the disulfide bridges of an F~ab~~2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F,, fragments.
Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions arid, if desired, the irnmunoglobulin 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 anothex approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (I985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments.
These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular 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')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
USA
90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.
See, Gruber et al., J. Immunol. 152:5368 (1994).
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 (FcyR), 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 (LT.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. Imrnunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et aI. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

Immunoconjugates The invention also pertains to irnmunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such irnmunoconjugates 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, PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for the production of radioconjugated antibodies.
Examples include 212Bi, isih i3lln, Soya and 186Re. ' 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 immunoliposoriies.
Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985);
Hwang et al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome.
See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX
protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX
protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Antibodies directed against a NOVX protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of a NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
In a given embodiment, antibodies specific to a NOVX protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as "Therapeutics").

An antibody specific for a NOVX protein of the invention (e.g., a monoclonal antibody or a polyclonal antibody) can be used to isolate a NOVX polypeptide by standard techniques, such as immunoaffinity, chromatography or irnmunoprecipitation. An antibody to a NOVX polypeptide can facilitate the purification of a natural NOVX
antigen from cells, or of a recombinantly produced NOVX antigen expressed in host cells. Moreover, such an anti-NOVX antibody can be used to detect the antigenic NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic NOVX protein. Antibodies directed against a NOVX protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include Izsh i3ih ass or 3H.
Antibody Therapeutics Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target.
Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question. In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
Pharmaceutical Compositions of Antibodies Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed.
(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA
technology.
See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(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 microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
ELISA Assay An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab)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 antitbody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample ifa vitro as well as ih vivo.
For example, ih vitro techniques for detection of an analyte mRNA include Northern hybridizations and ih situ hybridizations. Ih vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, irnmunoprecipitations, and irnmunofluorescence. 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, ira 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 episomal mammalian vectors). Other vectors (e.g., non-episomal rnamrnalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) in a manner that allows for expression of the nucleotide sequence (e.g., in an ih vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GE1~1E E~~SSIbN TECHNOLOGY: METHODS IN
ENZYMOLOGY 155, Academic Press, San Diego, Cali~ (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 proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX
proteins can be expressed in bacterial cells such as Esclaerichia 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 ifz vitro, for example using T7 promoter regulatory sequences and T7 polymerise.
Expression of proteins in prokaryotes is most often carned out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL
(New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Ge~ze 69:301-315) and pET l 1d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990) 60-89).
One strategy to maximize recombinant protein expression in E, coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS 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 DNA synthesis 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. Gehe 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Cali~), and pick (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. hirology 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 (Kaufinan, 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 CLONn~IG: 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. Immuhol.
43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;
Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurohlament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Scierace 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gauss, 1990. Science 249:
374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev.
3:
537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host Bells 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 transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method furfiher comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function andlor 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 manunal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences, I.e., any one of SEQ ID N0:2n-1, wherein h is an integer between 1 and 141, 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 transgenc-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of any one of SEQ ID NO:2n-1, wherein n is an integer between 1 and 141), 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 N0:2n-1, wherein . n is an integer between 1 and 141, 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. T'he additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA
(both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the 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. Opin.
Bioteehraol. 2:
823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO
92/0968;
and WO 93/04169.

In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad.
Sci. ZISA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Scietace 251:1351-1355.
If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilinut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable Garner" is intended to include any and all solvents, dispersion 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 Garners or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5%
human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (z.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 bisulflte;
chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Creinophor ELTM (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 arid 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, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active 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 carrier.
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 carrier 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 through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocornpatible 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 Garners. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical Garner.
The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci.
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 pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in a NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release);
obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX
protein activity.
The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a NOVX
protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; 'synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Araticarccer 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. Srnall molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
PYOC. 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. Artgew. Chem.
Irrt. Ed. Ehgl. 33:
2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J.
ll~Ied. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Iioughten, 1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. NatuYe 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Aead. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell.
Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with lzsh sss~ i4C~ 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 fornl of NOVX
protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX
protein, wherein determining the ability of the test compound to interact with a NOVX
protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX
protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with a NOVX
target molecule. As used herein, a "target molecule" is a molecule with which a NOVX
protein binds or interacts in nature, for example, a molecule on the 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., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX
protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
In still another embodiment, 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 can be accomplished, for example, by determining the ability of the NOVX protein to bind to a NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX
protein can be accomplished by determining the ability of the NOVX protein further modulate a NOVX
target molecule. For example, the catalytic/enzyrnatic activity of the target molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically-active portion thereof with a known compound which binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of a NOVX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane bound form of NOVX protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-100, Triton~ X-114, Thesit~, Isotridecypoly(ethylene glycol ether)n, N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX
protein, or interaction of NOVX protein with a target molecule in the presence and absence of a ~ candidate compound, can be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX
mRNA or protein in the cell is determined. The level of expression of NOVX
mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX
mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression.
The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chern.
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 identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX
activity. Such NOVX-binding proteins are also involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX
pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX
is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, ih 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 SEQ ID N0:2n-l, wherein h is an integer between 1 and 141, 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 axon 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. Scieraee 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 be identified individually.
The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES:
A MANUAL of BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDBLIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance 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 confirni the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA
markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).

Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX
sequences of the invention uniquely represent portions of the human genome.
Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If coding sequences, such as those of SEQ ID
N0:2n-1, wherein n is an integer between 1 and 141, are used, a more appropriate number of primers for positive individual identification would be S00-2,000.
Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX
protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX
expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, ~4 Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in a NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the~individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA
or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX
mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX
nucleic acid, such as the nucleic acid of SEQ ID N0:2n-1, wherein h is an integer between 1 and 141, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
~5 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')z) 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 ih vitro as well as in vivo.
For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in 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, ira vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody.
For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mltNA 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 adriiinistered the agent to treat a disorder associated with aberrant NOVX
expression or activity).
The methods of the invention can also be used to detect genetic lesions in a NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from a NOVX gene; (ii) an addition of one or more nucleotides to a NOVX gene; (iii) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate post-translational modification of a NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX
gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
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 amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878);
transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTechnology 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 1 S 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 NOVA 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. US'A 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. Biotechhiques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT
International.Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chronaatog~aphy 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechhol. 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, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc.
Natl. Acad. Sci. USA 85: 4397; Saleeba, et al., 1992. Methods Enzymol. 217:
286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. CaYCinogenesis 15: 1657-1662.
According to an exemplary embodiment, a probe based on a NOVX sequence, e.g., a wild-type NOVX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type , nucleic acids. See, e.g., Orita, et al., 1989. P~oc. Natl. Acad. Sci. USA: 86:
2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Gefaet. Araal. Tech. Appl. 9:
73-79.
Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA
(rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject.method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility.
See, e.g., Keen, et al., 1991. Trends Geraet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by of 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. Che~z. 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, allele specific amplification technology that depends on selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce 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. lllol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which rnay 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 but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.
In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype.
Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX

nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.
See e.g., Eichelbaum,1996. Clin. Exp. Plzarmacol. 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 polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and cytochrome pregnancy zone protein precursor enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizes (EM) and poor metabolizes (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identifted 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 ox drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring of Effects Dwring Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity ofNOVX (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 NOVA and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. 'Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the Levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i. e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.

In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidornimetic, 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, mltNA, 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, mltNA, 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 but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.
These methods of treatment will be discussed more fully, below.
Diseases and Disorders Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i. e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i. e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. 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 iyz 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.) andlor hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, ih situ hybridization, and the like).
Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX
activity.
Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections.
Therapeutic Methods Another aspect of the invention pertains to methods of modulating NOVX
expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate Iigand 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 in vitro (e.g., by culturing the cell with the agent) or, alternatively, iu vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect.
One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders).
Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).

Determination of the Biological Effect of the Therapeutic In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
In various specific embodiments, i~a 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 ih vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.
As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from diseases, disorders, conditions and the like, including but not limited to those listed herein.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A
further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which irnmunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES
Example A: Polynucleotide and Polypeptide Sequences, and Homology Data Example 1.
The NOVl clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 1A.

SEQ ID N0: 4 '257 as ~MW at 28672.2kD
....x......._._.....________.,.___._-.__--__-___-_ _-..____ . _.

otein Sequence SAPRRQDSEDHSSDMFNYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGC
KNSYRSEEACMLRCFRQQENPPLPLGSKVVVLAGLFVMVLILFLGASMVYLIRVA
~QERALRTVWSSGDDKEQLVKNTYVL
Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 1B.
Table 1B. Comparison of NOVla against NOVlb.
Protein Sequence NOVla Residues/ Identities/
Match Residues Similarities for the Matched Region NOVlb 5..256 249/257 (96%) 1..257 251/257 (96%) Further analysis of the NOVl a protein yielded the following properties shown in Table 1C.
Table 1C. Protein Sequence Properties NOVla PSort 0.8705 probability located in mitochondrial inner membrane;
analysis: 0.6000 probability located in plasma membrane; 0.4983 probability located in mitochondrial intermembrane space;
0.4000 probability located in Golgi body SignalP Cleavage site between residues 32 and 33 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 1D.
Table 1D. Geneseq Results for NOVla NOVla Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expeet Identifier [Patent #, Date] Match for Value the Residues MatchedRegion ABP41951 Human ovarian antigen 3..256 252/254(99%)e-148 HDABR73, SEQ ID N0:3083 17..270 253/254(99%) -Homo Sapiens, 270 aa.

[W0200200677-A1, 03-JAN-2002]

AAB43821 Human cancer associated 3..256 252/254(99%)e-148 protein sequence SEQ 17..270 253/254(99%) ID

N0:1266 - Homo Sapiens, aa. [W0200055350-A1, 2000]

AA017719 Human kunitz type protease5..256 250/252 (99%) e-148 inhibitor bikunin - Homo 1..252 251j252 (99%) Sapiens, 252 aa. [W09957274-A1, 11-NOV-1999]

AAB14187 Human placental bikunin 5..256 250/252 (99%) e-148 protein # 5 - Homo Sapiens, 1..252 251/252 (99%) 252 aa. [W0200037099-A2, 29-JUN-2000]

AAW70286 Human tissue factor pathway5..256 250/252 (99%) e-148 inhibitor-3 (TFPI-3) - Homo 1..252 251/252 (99%) Sapiens, 252 aa. [W09833920-A2, 06-AUG-1998]

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

Table 1E. Public BLASTP
Results for NOYla Identities/

Protein NOVla Similarities Residues/ Expect AccessionProtein/Organism/Length Match . for Value the Number Residues Matched Portion 043291 Kunitz-type protease inhibitor5..256 250/252(99%)e-147 2 precursor (Hepatocyte 1..252 251/252(99%) growth factor activator inhibitor type 2) (HAI-2) (Placental bikunin) - Homo Sapiens (Human), 252 aa.

Q9WU03 Kunitz-type protease inhibitor5..256 177/252(70%)e-102 2 precursor (Hepatocyte 1..252 202/252(79%) growth factor activator inhibitor type 2) (HAI-2) - Mus musculus (Mouse), 252 aa.

JG0185 hepatocyte growth factor 5..256 177/252(70%)e-102 activator inhibitor type 1..252 201/252(79%) mouse, 252 aa.

AAH03431Serine protease inhibitor,95..256 112/162(69%)3e-60 Kunitz type 2 - Mus musculus34..195 129/162(79%) (Mouse), 195 aa.

Q9D8Q8 Serine protease inhibitor,95..256 112/162(69%)3e-60 kunitz type 2 - Mus musculus34..195 129j162(79%) (Mouse), 195 aa.

S PFam analysis predicts that the NOV 1 a protein contains the domains shown in the Table 1F.
Table 1F. Domain Analysis of NOVla Pfam Domain~NOVla Match Region~Identities/ Expect Value Similarities for the Matched Region Kunitz BPTI,42..92 24/62 (39%) 9.7e-28 45/62 (73%) Kunit~ BPTI 137.,187 22/62 (35%) 2.6e-22 39/62 (63%) Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 2A.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 2B.
Table 2B. Comparison of NOV2a against NOV2b.
Protein Sequence NOV2a Residues/ Identities/
Match Residues Similarities for the Matched Region NOV2b 1..267 239/267 (89%) 1..239 239/267 (89%) Further analysis of the NOV2a protein yielded the following properties shown in Table 2C.
Table 2C. Protein Sequence Properties NOV2a PSort 0.6400 probability located in plasma membrane; 0.4600 analysis: probability located in Golgi body; 0,3700 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen) SignalP Cleavage site between residues 37 and 38 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/Similarities Expeet Identifier[Patent #~ Date] Mateh for Value the ResiduesMatchedRegion _........... ~"~",~", ~

AAM23963 Human EST encoded protein1..267 ~266/267(99%)e-157 SEQ

TD NO: 1488 - Homo Sapiens,1..267 267/267(99%) 267 an. [W0200154477-A2, AUG-2001]

AAW05732 Human metastasis tumour 2..267 266/267(99%)e-157 suppressor gene KAI1 product1..267 267/267{99%) [W09634117-A1, 31-OCT-1996]

ABB57295 Mouse ischaemic condition1..267 203/267 (76%) e-120 related protein sequence SEQ 1..266 230/267 (86%) ID N0:828 - Mus musculus, 266 aa. [W0200188188-A2, 22-NOV-2001]

AAB58792 Breast and ovarian cancer1..117 110/117 (94%) 4e-56 associated antigen protein 69..185 112/117 (95%) sequence SEQ ID 500 - Homo sapiens, 198 aa.

[W0200055173-A1, 21-SEP-2000]

AAG00436 Human secreted protein, 46..130 84/85 (98%) 5e-41 SEQ

ID NO: 4517 - Homo sapiens, 15..99 85/85 (99%) 99 aa. [EP1033401-A2, 06-SEP-_... ... . .......... ...... ................... ... . ......... ....
..2.~Ø..~ 1 . ........... . . .... . .. ........ ..
.... .. .... . ...~........ .......... .......... ......
........... . . .. ........ . . . ......... . .........
. ... ...
.

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/Similarities Expect Number Match for MatchedValue the Residues Portion AAH00726 Kangai 1 (suppression 1..267 267/267(100%)e-157 of ~

tumorigenicity 6, prostate,1..267 267/267(100%) CD82 antigen (R2 leukocyte antigen, antigen detected by monoclonal and antibody IA4)) - Homo sapiens (Human), as.

P27701 CD82 antigen (Inducible 1..267 266/267(99%) e-157 membrane protein R2) 1..267 267/267(99%) (C33 antigen) (IA4) (Metastasis suppressor Kangai 1) (Suppressor o tumorigenicity-6) - Homo sapiens (Human), 267 aa.

P40237 CD82 antigen (Inducible 1..267 203/267(76%) e-119 membrane protein R2) 1..266 230/267(86%) (C33 antigen) (IA4) - Mus musculus (Mouse), 266 aa.

070352 CD82 antigen (Metastasis1..267 202/267(75%) e-117 suppressor homology - 1..266 226/267(83%) Rattus norvegicus (Rat), 266 aa.

P11049 Leukocyte antigen CD37 4..267 99/276 2e-45 - Homo (35%) Sapiens (Human), 281 6..280 159/276(56%) aa.

PFam analysis predicts that the NOV2a protein contains the domains shown in the Table 2F.
Table 2F. Domain Analysis of NOV2a Identities/
Pfam Domain NOV2a Match Region: Similarities Expect Value for the Matched Region transmembrane4 10..256 102/270 (38%) 2.6e-96 221/270 (82%) Example 3.

The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 3B.
Table 3B. Comparison of NOV3a against NOV3b.
Protein Sequence NOV3a Residues/ Identities/
Match Residues Similarities for the Matched Region NOV3b 1..161 161/l85 (87%) 1..185 161/185 (87%) Further analysis of the NOV3a protein yielded the following properties shown in Table 3C.
Table 3C. Protein Sequence Properties NOV3a PSort 0.6000 probability located in plasma membrane; 0.4000 analysis: probability located in Golgi body; 0.3000 probability located in endoplasmic reticulum (membrane); 0.0300 probability located in mitochondrial inner membrane SignalP Cleavage site between residues 69 and 70 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 3D.
Table 3D. Geneseq Results for NOV3a NOV3a Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect Identifier[Patent #~ Date] Match for the MatchedValue ResiduesRegion AAM93733 Human polypeptide, SEQ 1..161 161/161 (100%)9e-86 ID

NO: 3697 - Homo Sapiens,1..161 161/161 (100%) aa. [EP1130094-A2, 05-SEP-2001]

ABG16996 Novel human diagnostic 47..133 24/89 (26%) 0.93 protein #16987 - Homo 280..36653/89 (58%) , sapiens, 2076 aa.

[W0200175067-A2, 11-OCT-2001]

ABP30247 Streptococcus polypeptide 64..114 23/56 (41%) 1.2 SEQ ID NO 9670 - 327..381 33/56 (58%) Streptococcus agalactiae, 401 aa. (W0200234771-A2, 02-MAY-2002]
ABP26074 Streptococcus polypeptide 64..114 23/56 (41%) 1.2 SEQ ID NO 1324 - 334..388 33/56 (58%) Streptococcus agalactiae, 408 aa. [W0200234771-A2, 02-MAY-2002]
ABB92972 Herbicidally active 58..123 22/68 (32%) 3.6 polypeptide SEQ ID NO 2183 - 175..236 37/68 (54%) Arabidopsis thaliana, 436 aa. [W0200210210-A2, 07-FEB-2002]
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 3E.

Table 3E. Public BLASTPResults for NOV3a NOV3a Identities/

Protein Residues/Similarities Expect AccessionProtein/Organism/LengthMatch for the MatchedValue Number ResiduesPortion Q96B96 Similar to hypothetical1..161 161/161 (100%)'3e-85 protein from clone 247961..161 161/161 (100%) -Homo sapiens (Human), aa.

000323 Hypothetical 17.6 kDa 1..161 159/161 (98%) 4e-84 protein - Homo sapiens 1..161 160/161 (98%) (Human), 161 aa.

Q922Z1 Similar to hypothetical1..158 112/159 (70%) 5e-57 protein from clone 247961..159 134/159 (83%) -Mus musculus (Mouse), aa.

P43932 Hypothetical protein 33..100 X19/68 (27%) 1.5 HI0056 :

- Haemophilus influenzae,168..22434/68 (49%) 237 aa.

Q9RZJ6 Hypothetical protein 33..96 20/67 (29%) 2.0 DRB0131 - Deinococcus 219..28535/67 (51%) radiodurans, 304 aa.

PFam analysis predicts that the NOV3a protein contains the domains shown in the Table 3F.
Table 3F. Domain Analysis of NOV3a Identities/
Pfam Domain NOV3a Match Region. Expect Value for the Matched Region No Significant Matches Found Example 4.
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.
Table 4A. NOV4 Sequence Anal SEQ ID N0: 13 ~ 1088 by fOV4a, GTGAGTTTACCCCTATGAGACTGTGAGAGGCCCGGGGCCTAC

Sequence GGCTTGGTTGTGAAGAGGCGGGGAAGCGGGTGTCCGGTCCCC
GCGCCTTCTTCCTGGACATGACCAACTGGAACCTACAAGCAGCAATTGGCGCCTATT
TGACTTTGAGAGCCCAAACATCAGTGTGCCCTCTATGTCCTTTGTTGAAGATGTCAC
TGTGAACATGGTGA
TGT
AACGCAGCAGCTGTCAT
CAAACAACTTATCAGTAGTGACTTACAGTAAGGGGCTCCATGGGCCTTACCC~
CCAGTCTTAAACGGGTGTCAGCAAAAAAAAP,AAAAAAAAA
Start: ATG atV162 ORF Stop: TAA at 1056 SEQ ID NO: 14 X298 as BMW at 32871.4kD
4a, MEGMDVDLDPELMQKFSCLGTTDKDVLISEFQRLLGFQLNPAGCAFFLDMTNW

tein Sequence YVGGDQFGHVNMVMVRSLEPQEIADVSVQMCSPSRAGMYQGQWRMCTATGLYY
VILSVEVGGLLGVTQQLSSFETEFNTQPHRKVEGNFNPFASPQKNRQSDENNL
SEFDSISKNTWAPAPDTWAPAPDQTEQDQNRLSQNSVNLSPSSHANNLSVVTY
GPYPFGQS
SEQ ID N0: 15 X735 by OV4b, G15160'8-02 DNA
equence CTACAAGCAGCAATTGGCGCCTATTATGACTTTGAGAGCC
ACCTCCGGATACTCAGTTTGTAAAAACATGGCGGATCCAGAATTCTGATGT
CCAGCAGTCACGCAAACAACTTATCAGTAGTGACTTACAGT
1~g Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 4B.
Table 413. Comparison of NOV4a against NOV4b.
NOV4a Residues/ Identities/
Protein Sequence Match Residues Similarities for the Matched Region NOV4b 171..298 128/128 (100%) 105..232 128/128 (100%) Further analysis of the NOV4a protein yielded the following properties shown in Table 4C.
Table 4C. Protein Sequence Properties NOV4a PSort 0.7000 probability located in plasma membrane; 0.3389 analysis: probability located in microbody (peroxisome); 0.2000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in mitochondrial inner membrane SignalP No Known Signal Sequence Predicted 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 Residues/Similarities Expect Identifier[Patent #. Date? Match for Matchedvalue the ResiduesRegion ABG40261 Human peptide encoded 172..287116/116(100%)2e-63 by genome-derived single 1..116 116/116(100%) exon probe SEQ ID 29926 -Homo sapiens, 116 aa.

[W0200186003-A2, 15-NOV-2001]

AAM18432 Peptide #4866 encoded 172..287116/116(100%)2e-63 by probe for measuring cervical1..116 116/116(100%) .....W~.._........_.."~,~"~,"~w. ............. ,.",~;~,.,x,~"~",",.
.~,~,"y,y, ...~ ,~,",,"
gene expression - Homo .
' Sapiens, 116 aa.

[W0200157278-A2, 09-AUG-2001]

AAM58143 Human brain expressed 172..287116/116(100%) 2e-63 single exon probe encoded protein1..116 116/116(100%) SEQ ID N0: 30248 - Homo Sapiens, 116 aa.

CW0200257275-A2, 09-AUG-2001]

ABB22766 Protein #4765 encoded 172..287116/116(i00%) 2e-63 by probe for measuring 2..116 116/116(100%) heart cell gene expression - Homo Sapiens, 116 aa.

[W0200157274-A2, 09-AUG-2001]

ABG15581 Novel human diagnostic 1..83 83/83 (100%) 7e-43 protein #15572 - Homo 44..126 83/83 (100%) Sapiens, 139 aa.

[W0200175067-A2, 11-OCT-2001]

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 NOV4a Protein NOV4a Identities/

Residues/Similarities Expect AccessionProtein/Organism/Length Match for MatchedValue Number the ResiduesPortion Q9BUR9 Hypothetical 32.9 kDa 1..298 298/298(100%) e-179 protein - Homo Sapiens 1..298 298/298(100%) (Human), 298 aa.

,Q96MG5 ~CDNA FLJ32402 fis, clone171..298128/l28(100%) 2e-71 'SKMLTS2000343 - Homo 105..232128/128(100%) Sapiens (Human), 232 aa.

Q9VX56 ~CG5445 protein (LD03052p)5..176 65/272 (37%) 8e-25 -Drosophila melanogaster 111..26394/172 (53%) (Fruit,fly), 303 aa.

Q9BL99 Hypothetical 28.4 kDa 8..179 52/184 (28%) 2e-16 ~

protein - Caenorhabditis4..186 92/184 (49%) elegans, 245 aa.

Q9SB64 'Hypothetical 76.2 kDa 77..180 38/110 (34%) 8e-13 ;protein - Arabidopsis 380..48758/110 (52%) thaliana (Mouse-ear cress), 704 aa.

PFam analysis predicts that the NOV4a protein contains the domains shown in the Table 4F.
Table 4F. Domain Analysis of NOV4a Identities/
Pfam Domain NOV4a Match Region Similarities Expect Value for the Matched Region No Significant Matches Found Example 5.
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide sequences axe shown in Table SA.

TCAGCTTCACAACTGTGTTGAAATTGCCTCAGCAATGGGACCTCAAGTGCT
TGATTTGGGGCATCACGGCTGTCACCCATGTCACTGCCATCCT
CCTTGT
ACTTTGGA
TGTAATCTGCAA
CTTCAAGGTTATACGGGTACTCAGTGTGGAGAATGCTCTACTGGTTTC
CAAGAATTTCAGGAGCACCTTGCCAACCATGTGCCTGCAACAACAACA
CGATCCAGAGTCCTGCAGCCGGGTAACAGGGGAGTGCCTTCGATGTTT
AAGATACTTTAAAGCTTACTAGTGCACTCAAAGTGAGCATGATAGTGAGACATGGTTT
~CTAAATGTGTAAAGAAAGTTTCTTTTATGTACTGTTGTTAATTAGTGCATTGAAACAG~
GGGTGGCCTTACAGGGGATGGAGTCAGCCTCTATCAAGGAATGAAAACCAAAAAAAGA
Start: ATG at 81 ~ ~ORF Stop: TAG at 3384 SEQ ID NO: 18 1101 as ~MW at 119568.2kD
5a, MQFQLTLFLHLGWLSYSKAQDDCNRUAC:HY'1"1't~yLLVUtttv'1'uLru~Sa't'~ULSxvS~nz tein Sequence RLDLEALFRFSHLILTFKTFRPAAMLVERSTDYGHNWKVFKYFAKDCATSFPNITSGQ
~AOGVGDIVCDSKYSDIEPSTGGEVVLKVLDPSFEIENPYSPYIQDLVTLTNLRINFTK
PNCERCKDFFQDAPWRP
DPALGSVAGQCLCKENVEGAKCDQCKPNHYGLSA
PCECDPDGTI
PAPGYFFAPLNFYLYEAEEATTLQGLAPLGSETFGQSPAVH
GFARVLPGAGLRFAVNNIPFPVDFTIAIHYETQSAADWTVQ
OSKP~SFALPAATRIMLLPTPICLEPDVOYSIDVYFSOPLQ
ESHAHSHVLVDSLGLIPQINSLENFCSKQDLDEYQLHNCVEIASAMGPQVLPGACERL
IISMSAKLHDGAVACKCHPQGSVGSSCSRLGGQCQCKPLVVGRCCDRCSTGSYDLGHH
GCHPCHCHPQGSKDTVCDQVTGQCPCHGEVSGRRCDRCLAGYFGFPSCHPCPCNRFAE
LCDPETGSCFNCGGFTTGRNCERCIDGYYGNPSSGQPCRPCLCPDDPSSNQYFAHSCY
QNLWSSDVICNCLQGYTGTQCGECSTGFYGNPRISGAPCQPCACNNNIDVTDPESCSR
VTGECLRCLHNTQGANCQLCKPGHYGSALNQTCRRCSCHASGVSPMECPPGGGACLCD
PVTGACPCLPNVTGLACDRCADGYWNLVPGRGCQSCDCDPRTSQSSHCDQARYFKAY
Further analysis of the NOVSa protein yielded the following properties shown in Table SB.
Table SB. Protein Sequence Properties NOVSa PSort 0.4500 probability located in cytoplasm; 0.3000 probability analysis: located in microbody (peroxisome); 0.1000 probability located in mitochondrial matrix space; 0.1000 probability located in lysosome (lumen) SignalP Cleavage site between residues 20 and 21 analysis:
A search of the 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 NOV5a Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect for Identifier[Patent #~ Date] Match the Matched Value Residues Region AAY15457 Human laminin beta 1..1094 1094/1094(100%)0.0 'protein - Homo sapiens,1..1094 1094/1094(100%) 1761 aa. [W09919348-A1, APR-1999]

AAY15459 SEQ ID 5 of W09919347 1..1101 1094/1105(99%) 0.0 - ~

Homo Sapiens, 1105 1..1105 1094/1105(99%) aa.

[W09919348-A1, 22-APR-1999]

AAM48896 Laminin protein - 23..1094 539/1089(49%) 0.0 Unidentified, 1786 30..1098 707/1089(64%) aa.

[W0200193897-A2, 13-DEC-2001]

ABB81591 Human laminin 10 second23..1094 539/1089(49%) 0.0 chain protein sequence9..1077 707/1089(64%) SEQ

ID N0:8 - Homo Sapiens, 1765 aa. [W0200250111-A2, 27-JUN-2002]

ABB81590 Human laminin 10 second23..1094 539/1089(49%) 0.0 chain protein sequence30..1098 707/1089(64%) SEQ

ID NO:6 - Homo Sapiens, 1786 aa. [W0200250111-A2, 27-JUN-2002]

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

Protein Residues/Similarities for Expect AccessionProtein/Organism/LengthMatch the Matched Value Number ResiduesPortion Q9Y6U6 WUGSC:H RG015P03.1 23..10931059/1071 (98%) 0.0 protein - Homo Sapiens (Human),1..1069 1061/1071 (98%) 1631 as (fragment).

Q9UHI2 Laminin beta 1 related 13..767 746/760 (98%) 0.0 protein - Homo Sapiens 1..760 747/760 (98%) (Human), 761 as (fragment).

057484 Laminin beta 2-like chain23..1094542/1084(50%) 0.0 -Gallus gallus (Chicken), 42..1103712/1084(65%) 1792 aa.

AAM61767 Laminin beta 1 - 21..1094537/1092(49%) 0.0 Brachydanio rerio 24..1095712/1092(65%) (Zebrafish) (Danio rerio), 1785 aa.

CAC17320 Sequence 15 from Patent23..1094539/1089(49%) 0.0 W00066730 - Homo Sapiens 9..1077 707/1089(64%) (Human), 1765 as ( fragment ) .

PFam analysis predicts that the NOVSa protein contains the domains shown in the Table SE.
Table SE. Domain Analysis of NOVSa Identities/

Pfam NOV5a Match Similarities Expect Domain Region Value for the Matched Region;

lamininNterm28..263 114/266 (43%) 6.8e-104 _ 181/266 (68%) lamininEGF 265..329 18/71 (25%) 1.5e-09 _ 48/71 (68%) lamininEGF 332..392 20/65 (31%) 4.8e-18 _ 48/65 (74%) lamininEGF 395..452 27/60 (45%) 4.5e-19 _ 45/60 (75%) lamininEGF 455..503 28/59 (47%) 1.7e-14 _ 39/59 (66%) lamininEGF 506..548 20/59 (34%) 0.00014 _ 30/59 (51%) lamininEGF 769..814 24/59 (41%) 4.5e-11 _ 36/59 (61%) lamininEGF 817..860 23/59 (39%) ~8e-14 _ 37/59 (63%) lamininEGF 863..908 25/59 (42%) 6.4e-09 _ 35/59 (59%) lamininEGF 911..967 16/62 (26%) 0.00078 _ 36/62 (58%) lamininEGF 970..1019 21/60 (35%) 1.4e-14 _ 38/60 (63%) Example 6.
The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.

Protein Sequence PGIPENGFRTPSGGVFFEGSVARFHCQDGFKLKGATKRLCLKHFNGTLGWIPSDNSI
VQEDCRIPQIEDAEIHNKTYRHGEKLIITCHEGFKIRYPDLHNMVSLCRDDGTWNNL
~ICQGCLRPLASSNGYVNISELQTSFPVGTVISYRCFPGFKLDGSAYLECLQNLIWSS
QYGEWFPSYQVYCIKSEQTWPSTHETLLTTWKIVAFTATSVLLVLLLVILARMFQTKF
KAHFPPRGPPRSSSSDPDFVVVDGVPVMLPSYDEAVSGGLSALGPGYMASVGQGCPLP
VDDQSPPAYPGSGDTDTGPGESETCDSVSGSSELLQSLYSPPRCQESTHPTSDNPDII
ASTAEEVASTSPGIDIADEIPLMEEDP
Further analysis of the NOV6a protein yielded the following properties shown in Table 6B.
Table 6B. Protein Sequence Properties NOV6a PSort 0.8000 probability located in mitochondrial inner membrane;
analysis: 0.7000 probability located in plasma membrane; 0.2000 probability located in endoplasmic reticulum (membrane); 0.0646 probability located in microbody (peroxisome) 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 6C.
Table 6C. Geneseq Results for NOV6a NOV6a Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect Identifier[Patent #~ Date] Match for MatchedYalue the ResiduesRegion AAB80234 Human PR0222 protein 106..536430/431(99%) 0.0 - Homo sapiens, 490 aa. 49..479 430/431(99%) [W0200104311-A1, 18-JAN-2001]

AAU12326 Human PR0222 polypeptide106..536430/431(99%) 0.0 sequence - Homo sapiens,49..479 430/431(99%) aa. [W0200140466-A2, 2001]

..
AAY13366 Amino acid sequence of 106..536430/431(99%) 0.0 protein PR0222 - Homo 49..479 430/431(99%) sapiens, 490 aa. [W09914328-A2, 2S-MAR-1999]

ABG26615 Novel human diagnostic 237..540299/353(84%) e-175 protein #26606 - Homo 1..353 300/353(84%) sapiens, 463 aa.

[W0200175067-A2, 11-OCT-2001]

ABB55790 Human polypeptide SEQ 106..298193/193(100%)1.e-117 TD NO

186 - Homo Sapiens, 49..241 193/193 (100%) 290 aa.

[US2001039335-A1, 08-NOV-' 2001]

In a BLAST seaxch of public sequence datbases, the NOV6a protein was found to have homology to the pxoteins shown in the BLASTP data in Table 6D.
"~"~...~.. ..................~...........~_.._........~m"~"~.~....~...~
.~.. .........................
... Results _......
Table 6D. Public BLASTP for NOV6a Protein NOV6a Identities/

Residues/Similarities Expect AccessionProtein/Organism/Length Match for MatchedValue Number the ResiduesPortion Q95K70 Hypothetical 43.3 kDa 7.57..549376/393(95%) 0.0 protein - Macaca 1..393 384/393(97%) fascicularis (Crab eating macaque) (Cynomolgus monkey), 393 aa, Q8VC43 Hypothetical 43.1 kDa 157..549356/393(90%) 0.0 protein - Mus musculus 1..393 372/393(94%) (Mouse), 393 aa.

Q9BSR0 Similar to hypothetical 106..298193/193(100%) e-117 protein FLJ10052 - Homo 49..241 193/193(100%) Sapiens (Human), 290 aa.

Q9NWG0 Hypothetical 26.1 kDa 106..242237/137(100%) 8e-82 protein - Homo Sapiens 49..185 137/137(100%) (Human), 236 aa.

Q92537 Hypothetical protein 299..49183/206(40%) 2e-30 KIAA0247 - Homo Sapiens 39..241 114/206(55%) (Human) , 303 aa.

PFam analysis predicts that the NOV6a pxotein contains the domains shown in the Table 6E.
Table 6E. Domain Analysis of NOV6a Identities/

Pfam DomainNOV6a Region SimilaritiesExpect Value Match for the Matched Region sushi 114..174 18/66 (27%)~ 7.2e-07 44/66 (67%) sushi 179..234 17/66 (26%) 1.5e-10 47/66 (71%) sushi 237..294 18/66 (27%) 6.4e-13 43/66 (65%) sushi 302..361 18/68 (26%) 4e-08 44/68 (65%) Example 7.
The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 7A.

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 7B.
Table 7B. Comparison of NOV7a against NOV7b.
Protein Sequence NOV7a Residues/ Identities/
Match Residues Similarities for the Matched Region NOV7b 1..351 349/351 (99%) 1..350 349/351 (99%) Further analysis of the NOV7a protein yielded the following properties shown in Table 7C.
Table 7C. Protein Sequence Properties NOV7a PSort 0.6500 probability located in plasma membrane; 0.4763 analysis: probability located in mitochondrial matrix space; 0.4500 probability located in cytoplasm; 0.2150 probability located in lysosome (lumen) SignalP Cleavage site between residues 12 and 13 analysis:
A search of the NOV7a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7D.
Table 7D. Geneseq Results for NOV7a NOV7a Identities/
Geneseq Protein/Organism/Length Residues/ Similarities Expect Identifier [Patent #, Date] Match for the Matched Value Residues Region AAB94678 Human protein sequence SEQ 65..351 287/287 (100%) e-169 ID N0:15628 - Homo Sapiens, 4..290 287/287 (100%) FEB-2001]
AAG45676 Arabidopsis thaliana protein 86..314 87/238 (36%) 2e-36 fragment SEQ ID NO: 57373 - 27..256 126/238 (52%) Arabidopsis thaliana, 310 aa. [EP1033405-A2, 06-SEP-2000]
AAG45675 Arabidopsis thaliana protein 86..314 87/238 (36%j 2e-36 fragment SEQ ID NO: 57372 - 105..334 126/238 (52%) Arabidopsis thaliana, 388 aa. [EP1033405-A2, 06-SEP-2000]
AAG45674 Arabidopsis thaliana protein 86..314 87/238 (36%) 2e-36 fragment SEQ ID NO: 57371 - 114..343 126/238 (52%) Arabidopsis thaliana, 397 aa. [EP1033405-A2, 06-SEP-2000]
AAG06884 Arabidopsis thaliana protein 86..314 87/238 (36%) 2e-36 fragment SEQ ID NO: 3823 - 27,.256 126/238 (52%) Arabidopsis thaliana, 310 aa. [EP1033405-A2, 06-SEP-2000]
~............ __...................... ... .....................
...................,.............
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 7E.
Table 7E. Public BLASTP Results for NOV7a Protein NOV7a Identities/

Residues/Similarities Expeet AccessionProtein/Organism/Length Match for MatchedValue Number the Residues Portion Q9CQ04 5730405M13Rik protein 1..351 300/351(85%) e-175 - Mus musculus (Mouse), 349 1..349 319/351(90%) aa.

Q9H8K6 CDNA FLJ13491 fis, clone65..351 287/287(100%) e-168 PZACE1004274 - Homo 4..290 287/287(100%) sapiens (Human) , 290 aa.

Q9DBJ4 1300006G11Rik protein 181..351 148/171(86%) 7e-85 (RIF~EN

cDNA 1300006611 gene) 1..171 157/171(91%) - Mus musculus (Mouse), 171 aa.

Q93W24 B1080D07.28 protein 140..324 84/199(42%) 3e-37 (P0507HO6.12 protein) 182..379 117/199(58%) -Ory~a sativa (Rice), 404 aa.

Q91,V19 Gb~AAB72I63.I (Unknown 86..314 82/239(34%) 1e-33 protein) - Arabidopsis 122..351 125/239(51%) thaliana (Mouse-ear cress), 394 aa.

PFam analysis predicts that the NOV7a protein contains the domains shown in the Table 7F.
Table 7F. Domain Analysis of NOV7a 2dentities/
Pfam Domain~NOV7a Match Region Similarities Expect Value for the Matched Region No Significant Matches Found Example 8.
The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.

Further analysis of the NOVBa protein yielded the following properties shown in Table 8B.
Table 8B. Protein Sequence Properties NOVBa PSort 0.6000 probability located in plasma membrane; 0.4000 analysis: probability located in Golgi body; 0.3000 probability located in endoplasmic reticulum (membrane); 0.2397 probability located in mitochondrial inner membrane SignalP Cleavage site between residues 1 and 2 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 8C.

Table 8C. Geneseq Results for NOVBa ' NOVBa Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect Identifier[Patent #. Date] Match for Value the ResiduesMatchedRegion AAB92881 Human protein sequence 1..323 290/323(89%) e-168 SEQ ID

N0:11479 - Homo Sapiens,1..291 290/323(89%) aa. [EP1074617-A2, 07-FEB-2001]
i AAM41733 Human polypeptide SEQ 1..323 290/323(89%) 2-168 ID NO

6664 - Homo Sapiens, 13..303 290/323(89%) 303 aa.

[W0200153312-A1, 26-JUL-2001]i AAM39947 Human polypeptide SEQ 1..323 290/323(89%) e-168 ID NO

3092 - Homo sapiens, W ..291 290/323(89%) 291 aa.

[W0200153312-A1, 26-JUL-2001]

ABB89884 Human polypeptide SEQ 1..323 288/323(89%) e-166 ID NO

2260 - Homo Sapiens, 1..291 288/323(89%) 291 aa.

[W0200190304-A2, 29-NOV-2001]' AAG74165 Human colon cancer antigen1..323 288/323(89%) e-166 protein SEQ ID N0:4929 13..303 288/323(89%) - Homo:

Sapiens, 303 aa.

[W0200122920-A2, 05-APR-2001]' In a BLAST search of public sequence datbases, the NOV8a protein was found to have homology to the proteins shown in the BLASTP data in Table 8D.
Table 8D. Public BLASTP Results for NOVBa Protein ~NOV8a Identities/ Expect Accession Protein/Organism/Length Residues/ Similarities Value Number Match for the ResiduesMatched Portion Q9NVV0 CDNA FLJ10493 fis, clone 1..323 290/323 (89%)e-167 NT2RP2000274 (Hypothetical1..291 290/323 (89%) 32.5 kDa protein) - Homo Sapiens (Human), 291 aa.

Q9DAV9 1600017F22Rik protein (RIKEN1..323 210/325 (64%)e-119 cDNA 1600017F22 gene) - 1..292 243/325 (74%) Mus musculus (Mouse), 292 aa.

Q9H6F2 CDNA: FLJ22328 fis, clone 7..321 121/324 (37%)9e-59 HRC05632 (Unknown) (Proteinx.1..297191/324 (58%) for MGC:3169) - Homo Sapiens (Human), 299 aa.

Q91WL2 Similar to hypothetical 7..321 117/323 (36%)5e-57 protein MGC3169 (Hypothetical.11..296187/323 (57%) 33.3 kDa protein) - Mus musculus (Mouse), 298 aa.

Q9VXG9 CG4239 protein (GH25683P) 14..27886/268 (32%)2e-33 -Drosophila melanogaster 15..249134/268 (49%) (Fruit fly), 276 aa.

PFam analysis predicts that the NOVBa protein contains the domains shown in the Table 8E.
Table 8E. Domain Analysis of NOVBa Identities/
Pfam Domain.NOV8a Match Region. Similarities Expect Value for the Matched Region No Significant Matches Found Example 9.
The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.

ATGCCAA.AAAATAGAAAAGCTATAGATATTTCAAAATAAAGAAGAA
ORF Start: ATG at 107 ~ ~ORF Stop: TAG at 779 SEQ ID N0: 28 224 aa~ MW at 24864.3kD
~.~~
a, MVAAVLLGLSWLCSPLGALVLDFNNIRSSADLHGARKGSQCLSDTDCNTRKFCLQ

ein Sequence GHPVQESQ~KRKPSIKKSQGRKGQEGESCLRTFDCGPGLCCARHFWTKICKPVLL
VCSRRGHKDTAQAPEIFQRCDCGPGLLCRSQLTSNRQHARLRVCQKIEKL
SEQ ID N0: 29 630 by ..y, ACCAGAAAGTTCTGCCTC
ORF Start: ATG at 67 ORF Stop: TAA at 586 __..
SEQ ID NO: 30 173 as MW at 19176.1kD
b, MVAAVLLGLSWLCSPLGALVLDFNNIRSSADLHGARKGSQCLSDTDCNTRKFCLQPR

ein Sequence PVLLEGQVCSRRGHKDTAQAPEIFQRCDCGPGLLCRSQLASNRQHARLRVCQKIEKL
iSEQ ID NO: 31 484 by JV9C, 05037558 bNA
~twlWtiACzcic.;AC:AGG'1'C:'1'GC:'1'CCAGAAGAGGGCATAA
TCTTCCAGCGTTGCGACTGTGGCCCTGGACTACTG
TCGGCAGCATGCTCGATTAAGAGTATGCCAAAAAA
Start: at 2 ORF Stop: end of sequenc~~,.,.~..~,..,.uv>
SEQ ID N0: 32 l61 as ,MW at 17937.4kD
UVyc, TGSLVLDFNNIRSSADLHGARKGSQCLSDTDCNTRKFCLQPRDEKPFCATCR

rotein Sequence DTAQAPEIFQRCDCGPGLLCRSQLASNRQHARLRVCQKIEKLLEG
SEQ ID NO: 33 541 by OV9d, equence TGTGCTGCCCTGGGACACTCTGTGTGAACGGACAAGAGGGAGAAA
1~4 .

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 9B.
~..... ..,_.~....................... ...................._........
_.....................................................
...............................................,~.. ....................r .u.....~.NOV9d, . .............
Table 9B. Comparison of NOV9a against NOV9b th o g NOV9a Residues/ Identities/
Protein Sequence~Match Residues Similarities for the Matched Region NOV9b 1..224 172/224 (76%) 1..173 172/224 (76%) NOV9c 17..224 155/208 (74%) 2..158 156/208 (74%) NOV9d 1..224 172/224 (76%) 5..177 172/224 (76%) Further analysis of the NOV9a protein yielded the following properties shown in Table 9C.
Table 9C. Protein Sequence Properties NOV9a PSort 0.7284 probability located in outside; 0.1000 probability analysis: located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in microbody (peroxisome) SignalP Cleavage site between residues 19 and 20 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 9D.
Table 9D. Geneseq Results for NOV9a NOV9a Identities/
Geneseq Protein/Organism/Length Residues/ Similarities Expect Identifier [Patent #. Date] Match for the Value Residues Matched Region AAY92075 Human DKR-4 - Homo Sapiens, 1..224 222/224 (99%) e-135 224 aa. [W0200018914-A2, 1..224 223/224(99%) APR-2000]

AAB08875Amino acid sequence of 1..224 222/224(99%) e-135 a human Dickkopf (Dkk)-4 1..224 223/224(99%) protein - Homo Sapiens, aa. [W0200052047-A2, 08-SEP-2000]

AAW73017Human cysteine-rich secreted1..224 222/224(99%) e-135 protein CRSP-2 - Homo 1,.224 223/224(99%) Sapiens, 224 aa. [W09846755-A1, 22-OCT-1998]

AAB66109Protein of the invention 34..221 84/199 (42%) 2e-37 #21 - Unidentified, 259 aa. 65..259 109/199(54%) [W0200078961-A1, 28-DEC-2000]

AAU29148Human PRO polypeptide 34..221 84/199 (42%) 2e-37 sequence #12S - Homo Sapiens,65..259 109/199(54%) 259 aa. [W0200168848-A2, SEP-2001]

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 9E.
Table 9E. Public BLASTP Results fox NOV9a NOV9a Identities/

Protein Similarities Residues/ Expect AccessionProtein/Organism/Length for the Match yalne Number Matched Residues portion Q9UBT3 Dickkopf related protein-41..224 222/224(99%)e-135 precursor (Dkk-4) (Dickkopf-1..224 223/224(99%) 4) (hDkk-4) - Homo Sapiens (Human), 224 aa.

Q8VEJ3 Similar to dickkopf (Xenopus1..221 166/221(75%)e-101 laevis) homolog 4 - Mus 1..221 185/221(83%) musculus (Mouse). 221 aa.

Q9UBU2 Dickkopf related protein-234..221 84/199(42%)7e-37 precursor (Dkk-2) (Dickkopf-65..259 109/299(54%) 2) (hDkk-2) - Homo Sapiens (Human), 259 aa.

Q9QYZ8 Dickkopf related protein-234..221 85/200(42%)9e-37 precursor (Dkk-2) (Dickkopf-65..259 109/200{54%) 2) (mDkk-2) - Mus musculus (Mouse), 259 aa.

Q9PWH3 Dickkopfl - Brachydanio 41..220 84/184(45%)1e-36 rerio (Zebrafish) (Zebra danio),68..239 105/184(56%) 240 aa.

PFam analysis predicts that the NOV9a protein contains the domains shown in the Table 9F.
Table 9F. Domain Analysis of NOV9a Identities/
Pfam Domain NOV9a Match Region Similarities Expect Value for the Matched Region No Significant Matches Found Example 10.
The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide Further analysis of the NOV 10a protein yielded the following properties shown in Table 10B.
Table 10B. Protein Sequence Properties NOVlOa PSort 0.8200 probability located in endoplasmic reticulum analysis: (membrane); 0.1900 probability located in plasma membrane;

sequences are shown in Table 10A.

0.1000 probability located inYendoplasmic reticulum (lumen);
0.1000 probability located in outside SignalP Cleavage site between residues 28 and 29 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 10C.

~ ~Table 10C. Geneseq Results for NOVlOa NOVlOa Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect Identifier[Patent #, Date] Match for MatchedValue the Residues Region AAU08753 Human insulin-like growth1..278 278/278(100%)e-169 factor binding protein-like1..278 278/278(100%) polypeptide #3 - Homo Sapiens, 278 as.

[W0200175064-A2, 11-OCT-2001]

AAE15654 Human growth factor 1..278 276/282(97%) e-164 binding protein-like protein, 1..282 276/282(97%) NOVS -Homo Sapiens, 282 as.

(W0200194416-A2, 13-DEC-2001]

AAU08755 Human insulin-like growth1..156 154/156(98%) 4e-93 factor binding protein-like1..156 155/156(98%) polypeptide #2 - Homo sapiens, 390 as.

[W0200175064-A2, 11-OCT-2001]
r ABG01683 Novel human diagnostic 1..156 154/156(98%) 4e-93 protein #1674 - Homo 1..156 155/156(98%) Sapiens, 390 as.

[W0200175067-A2, 11-OCT-2001]

AAR79102 Prostaglandin I2 (PGI2)11..262 115/263(43%) 4e-59 prodn. promoter - Homo 16..267 141/263(52%) Sapiens, 282 as. [W09429448-A, 22-DEC-1994]

In a BLAST search of public sequence datbases, the NOVlOa protein was found to have homology to the proteins shown in the BLASTP data in Table l OD.
Table 10D. Public BLASTP Results for NOVlOa Protein protein/Organism/Length NOVlOa Identities/ Expect Value Number Match for the Residues Matched Portion Q8WX77 BA113024.1 (similar to 1..278 278/278(100%)e-169 insulin-like growth factor1..278 278/278(100%)' binding protein) - Homo Sapiens (Human), 278 aa.

BAA21725IGFBP-LIKE PROTEIN - 1..276 212/276(76%)e-128 Mus musculus (Mouse), 270 1..268 234/276(83%) aa.

Q07822 MAC25 protein - Homo 11..262 115/263(43%),1e-58 Sapiens (Human), 277 aa. 16..267 141/263(52%) Q16270 Insulin-like growth factor11..262 115/263(43%)1e-58 binding protein 7 precursor16..267 141/263(52%) (IGFBP-7) (IBP- 7) (IGF-binding protein 7) (MAC25 protein) (Prostacyclin-stimulating factor) (PGI2-stimulating factor) -Homo Sapiens (Human), 282 aa.

Q61581 Mac25 protein - Mus musculus11..262 114/263(43%)5e-57 (Mouse), 281 aa. 15..266 140/263(52%) PFam analysis predicts that the NOVlOa protein contains the domains shown in the Table 10E.
Table 10E. Domain Analysis of NOVlOa Identities/
Pfam DomainiNOVlOa Match Region Similarities Expect Value for the Matched Region ka~al 91..151 18/63 (29%) 7.5e-05 45/63 (71%) ig 169..245 16/80 (20%) 5.4e-08 59/80 (74%) Example 11.
The NOV 11 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11A.

Further analysis of the NOV 11 a protein yielded the following properties shown in Table 11B.
Table 11B. Protein Sequence Properties NOVlIa PSort 0.5947 probability located in outside; 0.1000 probability analysis: 'located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 ;probability located in microbody (peroxisome) SignalP Cleavage site between residues 27 and 28 ' 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 11C.
Table 11C. Geneseq Results for NOVlla NOVlla Identities/
Geneseq Protein/Organism/Length Residues/ Similarities Expect Identifier [Patent #, Date] Match for the Value Residues. Matched Region AAY41496 Fragment of human secreted 40..334 235/303 (77%) e-120 protein encoded by gene 70 - 77..368 240/303 (78%) Homo Sapiens, 368 aa.
[W09947540-A1, 23-SEP-1999]

AAB07469 A human leucine-rich repeat9..290 93/284 (32%) 2e-28 protein designated Zlrr3 - 14..286126/284(43%) Homo Sapiens, 298 aa.

[W0200042184-A1, 20-JUL-2000]

AAU12198 Human PR01341 polypeptideX43..29085/250 (34%) 9e-28 sequence - Homo Sapiens, 281 :21..269116/250(46%) aa. [W0200140466-A2, 07-JUN-2001]

AAW96707 Protein sequence of the 34..23773/204 (35%) 8e-27 specification - Homo sapiens,~ 273..472107/204(51%) 1534 aa. [JP11018777-A, 26-JAN-1999]

AAW96706 Protein sequence of the 34..23773/204 (35%) 8e-27 specification - Homo sapiens,' 247..446107/204(51%) 1508 aa. [JP11018777-A, 26-JAN-1999]

In a BLAST search of public sequence datbases, the NOV1 la protein was found to have homology to the proteins shown in the BLASTP data in Table 11D.
Table lID. Public BLASTP Results for NOVlla Identities/

Protein NOVlla Similarities Residues/ Expect AccessionProtein/Organism/Length for the Match Value Number Matehed Residues Portion Q91W20 Unknown (Protein for 1..332 219/332(65%) e-112 MGC:6965) (Hypothetical 1..328 235/332(69%) 35.7 kDa protein) - Mus musculus (Mouse), 331 aa.

Q96B32 Hypothetical 35.0 kDa 62..285 81/226(35%) 6e-27 protein - Homo Sapiens 70..294 108/226(46%) (Human), 317 as (fragment).

BAA32465 MEGF4 - Homo Sapiens 34.,237 73/204(35%) 2e-26 (Human), 1618 as (fragment).357..556 107/204(51%) 075093 Slit-1 protein - Homo 34..237 73/204(35%) 2e-26 Sapiens (Human), 1534 273..472 107/204(51%) aa.

Q9WVB5 SLIT1 - Mus musculus 30..237 72/208(34%) 4e-26 (Mouse), 1531 aa, 269..472 109/208(51%) , PFam analysis predicts that the NOV1 la protein contains the domains shown in the.
Table 11E.
Table 11E. Domain Analysis of NOVlla Identities/
Pfam DomainNOVlla Match Region Similarities Expect Value .
for the Matched Region LRRNT 42..70 13/31 (42%) 0.86 20/31 (65%) LRR 96 . .119 9/25 i3~~) 0 5 . _.._ ~ (64%) LRR 120..143 11/25 (44%) 0.043 18/25 (72%) LRR X144..167 10/25 (40%) 0.33 17/25 (68%) LRRCT 201..254 18/55 (33%) 0.0078 30/55 (55%) Example 12.
The NOV12 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 12A.

Sequence comparison of the above protein sequences yields the following sequence 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 __._..__............._ i...-212 162/212 (76%) 1..163 162/212 (7,6%) Further analysis of the NOV 12a protein yielded the following properties shown in Table 12C.
Table 12C. Protein Sequence Properties NOVl2a PSort 0.6568 probability located in outside; 0.1000 probability analysis: located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in lysosome (lumen) SignalP Cleavage site between residues 23,and 24 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.

Table 12D. Geneseq Results for NOVl2a NOVl2a Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect Identifier[Patent #, Date1 Match for Value the Residues MatchedRegion ABP61861 Human polypeptide SEQ 1..212 211/212(99%) e-126 ID NO

215 - Homo Sapiens, 1..212 211/212(99%) 212 aa.

[US2002065394-A1, 30-MAY-2002]

AAM93517 Human polypeptide, SEQ 1..212 211/212(99%) e-126 ID

NO: 3243 - Homo Sapiens, 212 1..212 211/212 (99%) aa. [EPII30094-A2, 05-SEP-2001]

AAY94302 Human corticosteroid 1..212 211/212 (99%) e-126 synthesis-associated protein 1..212 211/212 (99%) - Homo Sapiens, 212 aa.

[W0200028027-A2, 18-MAY-2000]

AAW73630 Human secreted protein 1..212 211/212 (99%) e-126 clone ej265_4 - Homo Sapiens, 212 1..212 211/212 (99%) aa. [W09855614-A2, 10-DEC-1998]

AAY12939 Amino acid sequence of 1..212 172/212 (81%) 5e-96 a human secreted peptide - 1..212 179/212 (84%) Homo Sapiens, 213 aa.

[W09911293-A1, 11-MAR-1999]

In a BLAST search of public sequence datbases, the NOVl2a protein was found to have homology to the proteins shown in the BLASTP data in Table 12E.
Table 12E. Public BLASTP Results for NOVI2a Identities/

Protein NOVl2a Similarities AccessionProtein/Organism/Length Residues/for the Expect Number Match Matched Value Residues Portion Q9HBJ0 PLAC1 (Placenta-specific1..212 21.1/212 e-I26 1) - (99%) Homo Sapiens (Human), 1..212 211/222 (99%) 212 aa.

Q9JI83 EPCS26 (PLAC1) (Placental1..171 104/171 (60%)1e-60 specific protein 1) - 1..171 134/171 (77%) Mus musculus (Mouse), 173 aa.

BAC04191 CDNA FLJ36198 fis, clone9..125 38/118 (32%)7e-17 TESTI2028242, weakly 5..122 70/I18 (59%) similar to Mus musculus EPCS26 mRNA -Homo Sapiens (Human), 158 aa.

Q925U0 Initiate factor 3 (Oocyte-7..122 34/27.7 (29%)6e-09 secreted protein 1 precursor)8..122 62/117 (52%) - Mus musculus (Mouse), aa.

BAC11848 Initiate factor 3 2 - 7..88 25/83 (30%) 3e-05 Mus musculus (Mouse), 92 8..89 46/83 (55%) aa.

PFam analysis predicts that the NOVl2a protein contains the domains shown in the Table 12F.
Table I2F. Domain Analysis of NOVl2a Pfam Domain~NOVl2a Match Region~Identities/ Expect Value Similarities for the Matched Region No Significant Matches Found Example 13.
The NOV13 clone was analyzed, and the nucleotide and encoded polypeptide Further analysis of the NOVl3a protein yielded the following properCies shown in Table 13B.
Table 13B. Protein Sequence Properties NOVl3a PSort 0.6113 probability located in mitochondrial inner membrane;
analysis: 0.6000 probability located in plasma membrane; 0.4387 probability located in mitochondrial intermembrane space;
0.4000 probability located in Golgi body sequences are shown in Table 13A.

SignalP No Known Signal Sequence Predicted analysis 11:
A search of the NOV 13a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 13C.

Table 13C. Geneseq Results for NOVl3a NOVl3a Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect Identifier[Patent #. Data] Match for the MatchedValue Residues Region AAB41574 Human ORFX ORF1338 144..323 180/180 (100%)e-100 polypeptide sequence 1..180 180/180 (100%) SEQ ID

N0:2676 - Homo Sapiens, aa. [W0200058473-A2, 2000]

ABG21481 Novel human diagnostic 233..306 52/74 (70%) 3e-18 protein #21472 - Homo 48..120 56/74 (75%) Sapiens, 507 aa.

[W0200175067-A2, 11-OCT-2001]

AAG64212 Murine HSP47 interacting11..53 23/52 (44%) 0.21 protein, #2 - Mus sp, 65..115 27/52 (51%) aa. [JP2001145493-A, 2001]

ABB53290 Human polypeptide #30 11..53 23/52 (44%) 0.27 - Homo Sapiens, 255 aa. 65..115 27/52 (51%) [W0200181363-A1, O1-NOV-' 2001]

ABG20114 Novel human diagnostic 7..61 22/55 (40%) 0.35 protein #20105 - Homo 441..494 26/55 (47%) Sapiens, 710 aa.

[W0200175067-A2, 11-OCT-2001]

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

Protein Residues/ Similarities Expect AccessionProtein/Organism/LengthMatch for the Matched: Value Number Residues Portion Q9D7D4 2310014H19Rik proteinMus 30..323 277/294 (94%)e-157 -musculus (Mouse), 280/294 (95%) 288 aa. 1..288 Q9D8S1 1810038N08Rik proteinMus 30..323 277/294 (94%)e-157 -musculus (Mouse) , 288 aa. y1. .288~~280/294(95%) Q8R3U0 Similar to RIKEN cDNA 144..323170/180(94%) 5e-91 1810038N08 gene - Mus 1..174 171/180(94%) musculus (Mouse), 174 aa.

T49501 hypothetical protein 19..302 87/354 (24%) '3e-17 B14D6.530 [imported] - 149..496148/354(41%) Neurospora crassa, 556 aa.

Q12042 P2558 protein (ORF YPL162C)49..246 63/227 (27%) 3e-16 Saccharomyces cerevisiae 3..224 111/227(48%) (Baker's yeast), 273 aa.

PFam analysis predicts that the NOVl3a protein contains the domains shown in the Table 13E.
Table 13E. Domain Analysis of NOVl3a Identities/
Pfam Domain:NOVl3a Match Region Similarities Expect Value for the Matched Region No Significant Matches Found Example 14.
The NOVl4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 14A.

Table 14B.
Table 14B. Protein Sequence Properties NOVl4a PSort 0.4600 probability located in plasma membrane; 0.1000 analysis: probability located in 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:
S
A search of the NOV 14a 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 NOYl4a NOVl4a Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect Identifier;[Patent #~ Date] Match for MatchedValue the Residues Region AAU09695 Human melanoma antigen 26..575 550/550(100%)0.0 gp100 - Homo Sapiens, 112..661 550/550(100%) aa. [W0200192294-A2, DEC-2001]

AAU84803 Human gp100 consensus 26..575 550/550(100%)0.0 sequence - Homo Sapiens,112..661 550/550(100%) Further analysis of the NOVl4a protein yielded the following properties shown in 29-NOV-2001]

AAU29003 Melanoma antigen cDNA2526..575 550/550 (100%) 0.0 -.Synthetic, 661 aa. 112..661550/550 (100%) [US6270778-B1, 07-AUG-2001]

AAB47540 Human melanoma antigen 26..575 550/550 (100%) 0.0 gp100 - Homo Sapiens, 661 112..661550/550 (100%) aa. [W0200170767-A2, 27-SEP-2001]

AAY31977 Human melanoma antigen 26..575 550/550 (100%) 0.0 gp100 - Homo Sapiens, 661 112..661550/550 (100%) aa. [W09947102-A2, 23-SEP-In a BLAST search of public sequence datbases, the NOVl4a protein was found to have homology to the proteins shown in the BLASTP data in Table 14D.
Table 14D. Public BLASTP Results for NOVl4a NOVl4a Identities/

Protein Similarities AccessionProtein/Organism/Length Residues/for Expect the Number Match Matched 'Value Residues portion P40967 Melanocyte protein Pmel 26..575 550/550(100%)0.0 precursor (Melanocyte 112..661 550/550(100%) lineage-specific antigen GP100) (Melanoma-associated antigen) (ME20M/ME20S) (ME20-M/ME20-S) (95 kDa melanocyte-specific secreted glycoprotein) - Homo Sapiens (Human), 661 aa.

CAC38954 Sequence 109 from Patent26..575 548/550(99%)0.0 W00130382 - synthetic 112,.661 548/550(99%) construct, 661 aa.

I38400 melanoma-associated ME2026..575 550/551(99%)0.0 antigen (me20m) - human,112..662 550/551(99%) aa.

A41234 melanocyte-specific protein26..575 549/557(98%)0.0 Pmel-17 precursor - human,112..668 549/557(98%) aa.

Q9CZB2 N/A - Mus musculus (Mouse),26..575 415/550(75%)0.0 626 aa. 111..626 448/550(81%) PFam analysis predicts that the NOVl4a protein contains the domains shown in the Table 14E.
Table 14E. Domain Analysis of NOVl4a Identities/
Pfam Domain~NOVl4a Match Region Similarities Expect Value for the Matched Region PKD 1131..215 26/99 (26%) 5.6e-08 61/99 (62%) Example 15.
The NOV15 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 15A.

SEQ ID N0: 48 X458 as BMW at 50069.3kD
. , .r,..L~rauL rv r y1 v ~o.w.LL.ut~.~tuirrr~r r riv.ur a r mr rJ o W
tuil~LHAtCIiY~ t'iLLla'"1'iSKLtS

otein Sequence FVPLELRVTAASGAPRYHRVTHINEVVLLDAPVGLVARLADESGHVVLRWLPPPETPM
TSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGF
WSAWSEPVSLLTPSDLDPLILTLSLILWILVLLTVLALLSHRRALKQKIWPGIPSPE
SEFEGLFTTHKGNFQLWLYQNDGCLWWSACTPFTEDPPAFLEVLSERCWGTMQAVEPG
TDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPWALCPELPPTPPHLKYLY
~LWSDSGISTDYSSGDSQGAQGGLSDGPYSSPYENSPIPAAEPLPPSYVACS
SEQ ID NO: 49 1733 by quence AACC
GCCTCCGGCGCTCCGCGA
CGCCCAGTGAGGACCTCCCAGGGCCATGGGCACTGTGCCCTGAGCTGC
Start: ATG at 145 ~ORF Stop: TAG at 1519 SEQ ID N0: 50 458 as ~MW at 50069.3kD
OVl5b, MDHLGASLWPQVGSLCLLLAGAAWAPPPNLPDPKFESKAALLAARGPEELLCFTERLE

rotein Sequence FVPLELRVTAASGAPRYHRVIHINEVVLLDAPVGLVARLADESGHVVLRWLPPPETPM
TSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGF
WSAWSEPVSLLTPSDLDPLILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPE
SEFEGLFTTHKGNFQLWLYQNDGCLWWSACTPFTEDPPAFLEVLSERCWGTMQAVEPG
~VVSDSGISTDYSSGDSQGAQGGLSDGPYSSPYENSPIPAAEPLPPSYVACS
ID N0: 51 1435 by uvl5c, equence AGTGCTTCT
CT
GCTTCTGGAGCGCCTGGTCGGAGCCTGTGTCGCTGCTGACGCCTAGCGACCT
CCTCATCCTGACGCTCTCCCTCATCCTCGTGGTCATCCTGGTGCTGCTGACC
rcrcTrrTCTCCCACCGCCGGGCTCTGAAGCAGAAGATCTGGCCTGGCATCC
CGAGCC
GTGGCTGTACCAGAATGATGGCTGCCTGTGGTGGAGC:c:c:c:'1'GCr~ccCCC:
GACCCACCTGCTTCCCTGGAAGTCCTCTCAGAGCGCTGCTGGGGGACGA
TGGAGCCGGGGACAGATGATGAGGGCCCCCTGCTGGAGCCAGTGGGCAG
CTACTCCAGCCCTTA
TATGACTCAGAGAACC
Start: ATG at 12 ~ ~ORF Stop: TAG at 1386 ID NO: 52 X458 as BMW at 49993.2kD
NOV 15 C , MDHLGASLWYQVGSLC:LLLAGHAWAYYYNLYLYttr'~~l~l~t~tuir~~Lm:r i c~rcLu Protein Sequence FVPLELRVTAASGAPRYHRVIHINEVVLLDAPVGLVARLADESGHVVLRWLPPPETPM
TSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRTRMAEPSFGGF
WSAWSEPVSLLTPSDLDPLILTLSLILWILVLLTVLALLSHRRALKQKIWPGIPSPE
SEFEGLFTTHKGNFQLWLYQNDGCLWWSPCTPFTEDPPASLEVLSERCWGTMQAVEPG
TDDEGPLLEPVGSEHAQDTYLVLDKWLLPRNPPSEDLPGPWALCPELPPTPPHLKYLY
LVVSDSGISTDYSSGDSQGAQGGLSDGPYSSPYENSPIPAAEPLPPSYVACS
ID NO: 53 1585 by Vl5d, quence TCC
CAGCTTTGAGTACACTAT

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 15B.
Table 15B. Comparison of NOVlSa against NOVlSb through NOVlSd.
NOVl5a Residues/ Identities/

ProteinSequence Similarities for the Matched Match Residues Region NOVlSb 1..458 458/458 (100%) 1..458 458/458 (100%) NOVl5c 1..458 454/458 (99%) 1..458 454/458 (99%) NOVl5d 1..458 450/508 (88%) 1..508 452/508 (88%) Further analysis of the NOVlSa protein yielded the following properties shown in Table 15C.
Table 15C. Protein Sequence Properties NOVlSa PSort 0.4600 probability located in plasma membrane; 0.1762 analysis: probability located in microbody (peroxisome); 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen) SignalP Cleavage site between residues 26 and 27 analysis : , .~ . ... . ..

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 15D.

Table 15D. Geneseq Results for NOVlSa NOVl5a Identities/

Geneseq 'Protein/Organism/LengthResidues/Similarities Expect Identifier[Patent #~ Data] Match for Value the ResiduesMatchedRegion AAR69503 Human erythropoietin 1..458 453/508(89%) 0.0 receptor - Homo Sapiens,1..508 454/508(89%) aa. [US5378808-A,'03-JAN-1995]

AAR70032 :Human erythropoietin 1..458 453/508(89%) 0.0 preceptor - Homo Sapiens,1..508 454/508(89%) aa. [W09505469-A, 23-FEB-1995]

AAR06512 EPO receptor - Homo Sapiens,1..458 453/508(89%) 0.0 508 aa. [W09008822-A, 1..508 454/508(89%) AUG-1990]

ABB09173 Human,erythropoietin 1..458 452/508(88%) 0.0 ~

receptor SEQ ID N0:5 1..508 453/508(88%) - Homo Sapiens, 508 aa.

[US2002031806-A1, 14-MAR-2002]

AAY44622 !Truncated human EpoR(t439)1..388 386/388(99%) 0.0 -Homo sapiens, 438 aa. 1..388 386/388(99%) [W09967360-A2, 29-DEC-1999]

In a BLAST search of public sequence datbases, the NOVlSa protein was found to have homology to the proteins shown in the BLASTP
data in Table 15E.

Table 15E. Public BLASTPResults for NOVlSa ~ NOVl5a Identities/

Protein Residues/Similarities Expect AccessionProtein/Organism/LengthMatch for MatchedValue the Number Residues Portion P19235 Erythropoietin receptor1..458 453/508(89%) 0.0 precursor (EPO-R) - 1..508 454/508(89%) Homo Sapiens (Human), 508 aa. I

Q9MYZ9 Erythropoietin receptor1..458 386/509(75%) 0.0 -Sus scrofa (Pig), 509 1..509 402/509(78%) aa.

P14753 Erythropoietin receptor1..458 375/508(73%) 0.0 precursor (EPO-R) - 1..507 397/508(77%) Mus musculus (Mouse), 507 aa.

AAU03953 Similar to erythropoietin 2..458374/507 (73%) 0.0 receptor - Mus musculus 1..506396/507 (77%) (Mouse), 506 as (fragment).

Q07303 Erythropoietin receptor 1..458371/508 (73%) 0.0 precursor (EPO-R) - Rattus 399/508 (78%) 1..507 norvegicus (Rat), 507 aa.

PFarn analysis predicts that the NOVlSa protein contains the domains shown in the Table 15F.
Table 15F. Domain Analysis of NOVlSa Identities/
Pfam Domain NOVl5a Match Region Similarities Expect Value for the Matched Region fn3 145..228 21/88 (24%) 0.00059 59/88 (67%) Example 16.
The NOV 16 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 16A.

CAGGCGCCCA
AGCGCCGAGATCCTGGCGGCCTACCGGCCCGCCGTGCACCCCCGCTAGCGC
. ......................... ~. .-.......... ............. . .. .........
....._~~.................. ............. ... _ . .. ....... .. p .
Start: ATG at 7 ORF Sto . TAG at 688 SEQ ID NO: 58 227 as MW at 25573.8kD
NOVl6b, MAETKLQLFVKASEDGESVGHCPSCQRLFMVLLLKGVPFTLTTVDTRRSPDVLKDF

Protein Sequence ALYQQLLRALARLDSYLRAPLEHELAGEPQLRESRRRFLDGDRLTLADCSLLPKLH
DTVCAHFRQAPIPAELRGVRRYLDSAMQEKEFKYTCPHSAEILAAYRPAVHPR
SEQ ID NO: 59 784 by Vl6c, CGGCCGCGTCGACGCGGCAGCTCCCACCA

TACACGTGTCCGCACAGCGCCGAGATCCTGGCGGCCTACCGGCCCGC
c'c'TAGCGCCCCACCCCGCGTCTGTCGCCCAATAAAGGCATCTTTGTC
ORF Start: ATG at 29 ~ORF Stop: TAG at 710 SEQ ID N0: 60 227 as ~MW at 25573.8kD
16c~,~~ MAETKLQLFVKASEDGESVGHCPSCQRLFMVLLLKGVPFTLTTVDTRRSPDVLK

tein Sequence ALYQQLLRALARLDSYLRAPLEHELAGEPQLRESRRRFLDGDRLTLADCSLLPK
DTVCAHFRQAPIPAELRGVRRYLDSAMQEKEFKYTCPHSAEILAAYRPAVHPR
SEQ ID NO: 61 ~ 751 by NOVl6d, Sequence TCAAGAACC
CCCGCCGCCGCTTCCTGGACGGCGACAGGCTCACGCTGGCCGACTGCAGCCTCCTGCC
CAAGCTGCACATCGTCGACACGGTGTGCGCGCACTTCCGCCAGGCGCCCATCCCCGCG
CCGCGTCTGTCGCCCAATAAAGGCATCTTTGTCGGGAAAAAA

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 16B.
Table 16B. Comparison of NOVl6a against NOVl6b through NOVl6e.
NOVl6a Residues/ Identities/

ProteinSequence Match ResiduesSimilarities for the Matched . Region ................_., ....
.~~,",,~"", .~ ............
...... .. ..

NOVl6b ,"~"~"~ ",,,~
1..189 189/189 (100%) 1..189 189/189 (100%) N0V16c 1..189 189/189 (100%) 1..189 189/189 (100%) NOVl6d 1..227 227/227 (100%) 1..227 227/227 (100%) NOVl6e 1..227 227/227 (100%) 1..227 227/227 (100%) Further analysis of the NOVl6a protein yielded the following properties shown in Table 16C.
Table 16C. Protein Sequence Properties NOVl6a PSort 0.9000 probability located in Golgi body; 0.7900 probability analysis: located in plasma membrane; 0.3000 probability located in microbody (peroxisome); 0.2000 probability located in endoplasmic reticulum (membrane) SignalP Cleavage site between residues 43 and 44 analysis ~ ,......
A search of the NOVl6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 16D.
Table 16D. Geneseq Results for NOVl6a NOVl6a Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect Identifier[Patent #, Date] Match for Value the Residues MatchedRegion AAW61550 Human chloride channel 1..227 226/236(95%)e-129 protein - Homo Sapiens,6..241 227/236(95%) aa. [W09830691-Al, 16-J'Uh-1998]

AAU23722 Novel human enzyme 20..189 162/179(90%)8e-87 polypeptide #808 - Homo6..184 163/179(90%) Sapiens, 222 aa.

[W0200155301-A2, 02-AUG-2001] , AAM40512 Human polypeptide SEQ 3..189 101/198(51%)6e-49 ID NO

5443 - Homo sapiens, 60..257 134/198(67%) 312 aa.

[W0200153312-A1, 26-~TUh-2001]

AAM38726 Human polypeptide SEQ 3..189 101/198(51%)6e-49 ID NO

1871 - Homo Sapiens, 71..268 134/198(67%) 308 aa.

[W0200153312-A1, 26-JUL-2001]

AAM79354 Human protein SEQ ID 3..189 101/198(51%)6e-49 - Homo Sapiens, 312 60..257 134/l98(67%) aa.

[W0200157190-A2, 09-AUG-2001]
:

14~

In a BLAST search of public sequence datbases, the NOVl6a protein was found to have homology to the proteins shown in the BLASTP
data in Table 16E.

Table 16E. Public BLASTPResults for NOVl6a Identities/

Protein NOVl6a Similarities AccessionProtein/Organism/Length Residues/for Expect the Match Value Matched Number Residues Portion 095833 Chloride intracellular 30..189 159/169(94%)4e-85 channel protein 3 - Homo1..169 160/169(94%) Sapiens (Human), 207 aa.

Q9D7P7 2300003G24Rik protein 30..189 143/169(84%)2e-76 - Mus musculus (Mouse), 207 1..169 149/169(87%) aa.

Q9ZOW7 Chloride intracellular 3..187 102/196(52%)3e-49 channel protein 4 16..211 133/196(67%) (Intracellular chloride ion channel protein P64H1) -Rattus norvegicus (Rat), aa.

Q9QYB1 Intracellular chloride 3..187 102/196(52%)5e-49 channel protein - Mus 16..211 133/196(67%) musculus (Mouse), 253 aa.

Q9Y696 Chloride intracellular 3..189 101/198(51%)2e-48 channel protein 4 16..213 134/198(67%) (Intracellular chloride ion channel protein p64H1) - Homo Sapiens (Human), 253 aa.

PFam analysis predicts that the NOV 16a protein contains the domains shown in the Table 16F.
Table 16F. Domain Analysis of NOVl6a Identities/
Pfam Domain NOVl6a Match Region Similarities Expect Value for the Matched Region ~.
No Significant Matches Found Example 17.
The NOV17 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 17A.
Table 17A.1VOV17 Sequence Analysis SEQ ID NO: 65 2400 by dOVl7a, GTGCGCGTTGGGGCGGCCGGCCAATGCCGGACCGCTTCGGCACCGCCCGCCCGATCCC

~G159015-O1 TCCACCCGTGGGCCGGCAATGGCGGGCGCAGTTTCGCTCTTGGGTGTGGTGGGGCTGC
DNA

Sequence TGCTTGTGTCTGCGCTGTCCGGGGTCCTAGGAGACCGCGCCAATCCCGACCTCCGGGC

ACACCCAGGGAACGCAGCCCACCCCGGCTCTGGAGCCACGGAACCCCGGCGGCGACCA

CCGCTCAAGGATCAACGCGAGCGGACCCGGGCCGGGTCGCTGCCTCTGGGGGCGCTGT

ACACCGCGGCCGTCGCGGCTTTTGTGCTGTACAAGTGTTTGCAGGGGAAAGATGAAAC

TGCGGTTCTCCACGAGGAGGCAAGCAAGCAGCAGCCACTGCAGTCAGAGCAACAGCTG

GCCCAGTTGACACAACAGCTGGCCCAGACAGAGCAGCACCTGAACAACCTGATGGCCC

AGCTGGACCCCCTTTTTGAGCGTGTGACTACTCTGGCTGGAGCCCAGCAGGAGCTTCT

GAACATGAAGCTATGGACCATCCACGAGCTGCTGCAAGATAGCAAGCCGGACAAGGAT

ATGGAGGCTTCAGAACCAGGTGAAGGCTCGGGAGGCGAGTCTGCTGGAGGTGGAGACA

AAGTCTCTGAAACTGGAACATTCCTGATCTCTCCCCACACAGAGGCCAGCAGACCTCT

TCCTGAGGACTTCTGTTTAAAGGAGGACGAGGAGGAGGTTGGTGACAGTCAGGCCTGG

GAGGAGCCCACAAACTGGAGCACAGAGACATGGAACCTAGCTACTTCCTGGGAGGTGG

GGCGGGGACTACGGAGAAGGTGCAGCCAGGCTGTGGCAAAGGGCCCCAGTCACAGCCT

TGGCTGGGAAGGAGGGACGACAGCTGAAGGTCGACTAAAACAAAGTCTGTTTTCATGA

TGGAGTGCTCCTGTGTGTTTTTTCGATCCTAGTTGGTTGTACACACCCATACTAGGTG

CCTAAGGACAACTGGGCCTTCTTGAAGAGCTGTCCTTATTAGGACAAAAAGAGGCTGC

CTTCCAGTGTGACAGCAGAGAAGATAGAGGGAGCTCCAGCTCTTTTCCTCGTATTCCT

GAGGCCACCAGCATGCCCGCGTTCAGGGCCCAAi~AATCCCTTTTCTCATAGCAAAACT

GAGACAGAAGGGTCTTTCCCAAAAAAAAGF~~AI~AAAAACTTTACTCAAATCCAGTGGA

AAAATAAATGATAGAAACTATACACAACATAAT~AATAGCCACATTTACAAAGCTGCAG

CCTTGATAAATGACGGGCCATGGACACAGCACAGAGCTTATCAGTCCCAAATCCCCTC

ATCTGTGTTAGGGGCTGGTTCATTTGAGGTTTAGTTGGGTTGGACTTGGTTTCCTGAT

TCTTCTTTTTTAATAAAATTTCTTAATTATTTTTTCTTAAATAGAGACAGGGTCTCAC

TCACTGTGTTGCCCAGGCTGGTCTTGAACTCCTGGGCTGGAATGATCCTGCCACCTCT

GCTTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGTGCCTGGCCGTGATTTTTAA

GAGTTGGTCAGATGATCTGGAGTAGCTTGGTCCAGGCAAACAGAAAGTGACCTTTGTC

AAATCATGAAGGGTTCTGTTTTGTTCAGTACTGAAGATTCCTTTGTACTCTTGGCTGT

GACCTATCCCTGAGGTATCCTGAGTTCTGGAATCTATAAGATTCCTCTAGTTTTTCTG

GCTGCTGATAGCCCAAGTCAGACTGTGGTACCAGCGTGACAGCTCCTCCTGGTCTGTG

GACATAAGCAGTAGCTTCTCATGAGGGAAGGACAGGTGTGAGCTGTTGATGGTCAGGG

CTGTTGGGACCTGTGTTTTCAGCCAAAGCTACGACGAGATTCTCATACTGCTGGAGCC

GTTGCAGAGGCAGAGGGAGCAGGTCCTGGAGCTGAAGGCCCCCAAACCCAGGGCGGCC

TTCCTGAAGCCGTACAAACCTCCGGAAACCTTTATTTTTCTTTAGCTGCTCCTGCAGG

GTGGTCTGGGACCTCTCTGAGTTGGCAGCAAATTGGTTATAGAGCTCCAAGTGGCGGC

AGAAGCCCTCCAGCCCTTGGCCCCAGCATCCTCCTTCCAGGTAGGGAAGCAGCTCCTG

GCTGGCGCCGTAGATGAGCTCCCAGGAGCCAAACAGGGCCTGGCGCTCAGGTGGTCGC

AGGGTCCCCTTGGCTTTCAGGATCCCCAAAAAGTACGTGGCCACCAGCCCCAGCTGTT

CTTGGTAGCGCCGCTCGGTCTCTAGCAGCTCCCGGGCGGTGCAGGCGCGTTTCCGCTC

CCAGCGGGCACGCTGCTCTTGCACCGGGCACCGCGAACCGGGGCAGGAGAGCTCCATG

CCCTGGCTGAGGGATCGACACT

ORF Start: ATG at 77 ORF
Stop: TGA at 926 . u.. . ~ SEQ ID NO: 66 v ~.... W
.y.~.83 a-"~ t 30494..7kD
.....
...
.
.
_....

_.._...... MAGAVSLLGWGLLLVSALSGVLGDRANPDLRAHPGNAAHPGSGATEPRRRPPLKDQR
.........
NOVl7a, rotein SequenceLAQTEQHLNNLMAQLDPLFERVTTLAGAQQELLNMKLWTIHELLQDSKPDKDMEASEP

GEGSGGESAGGGDKVSETGTFLISPHTEASRPLPEDFCLKEDEEEVGDSQAWEEPTNW

STETWNLATSWEVGRGLRRRCSQAVAKGPSHSLGWEGGTTAEGRLKQSLFS

SEQ ID NO: 67 1449 by OVl7b, GGTGAGAAAGTTGGTGGCGTGAGATTAAfiAAAACCGTTTTCGGGCATAACTTTCTAAG

DNA

equence AAATACAGGAAAGCTAGAATGACACTATCTTATGCAAATATGGTCTGGCCCCGCCCTA

CGGGGAGTGGGCGTGGCCTCCCCGGAGCCGGCCGGCCTGCTCGCGTGCGCGTGCGCGT

TGGGGCGGCCGGCCAATGCCGGACCGCTTCGGCACCGCCCGCCCGATCCCTCCACCCG

TGGGCCGGCAATGGCGGGCGCAGTTTCGCTCTTGGGTGTGGTGGGGCTGCTGCTTGTG

TCTGCGCTGTCCGGGGTCCTAGGAGACCGCGCCAATCCCGACCTCCGGGCACACCCAG

GTAACGCAGCCCACCCCGGCTCTGGAGCCACGGAACCCCGGCGGCGACCACCGCTCAA

GGATCAACGCGAGCGGACCCGGGCCGGGTCGCTGCCTCTGGGGGCGCTGTACACCGCG

TCCACGAGGAGGCAAGCAAGCAGCAGCCACTGCAGTCAGAGCAACAGCTGGCCCAGTT

GACACAACAGCTGGCCCAGACAGAGCAGCACCTGAACAACCTGATGGCCCAGCTGGAC

CCCCTTTTTGAGCGGGTGACTACTCTGGCTGGAGCCCAGCAGGAGCTTCTGAACATGA

AGCTATGGACCATCCACGAGCTGCTGCAAGATAGCAAGCCGGACAAGGATATGGAGGC

TTCAGAACCAGGTGAAGGCTCGGGAGGCGAGTCTGCTGGAGGTGGAGACAAAGTCTCT

GAAACTGGAACATTCCTGATCTCTCCCCACACAGAGGCCAGCAGACCTCTTCCTGAGG

ACTTCTGTTTAAAGGAGGACGAGGAGGAGATTGGTGACAGTCAGGCCTGGGAGGAGCC

CTACGGAGAAGGTGCAGCCAGGCTGTGGCAAAGGGCCCCAGTCACAGCCTTGGCTGGG

AAGGAGGGACGACAGCTGAAGGTCGACTAAAACAAAGTCTGTTTTCATGATGGAGTGC

TCCTGTGTGTTTTTTCGATCCTAGTTGGTTGTACACACCCATACTAGGTGCCTAAGGA

CAACTGGGCCTTCTTGAAGAGCTGTCCTTATTAGGACAAAAAGAGGCTGCCTTCCAGT

CAGCATGCCCGCGTTCAGGGCCCAAAAATCCCTTTTCTCATAGCAAAACTGAGACAGA

AGGGTCTTTCCCF, ~~IAAAGAAAAAAAACTTTACTCAAATCCAGTGGAAAAATAAA

ORF Start: ATG at 148 ORF Stop: TGA at 1150 SEQ ID NO: 68 334 as MW at 35589.5kD

OVl7b, MQIWSGPALRGVGVASPEPAGLLACACALGRPANAGPLRHRPPDPSTRGPAMAGAVSL

rotein SequenceLPLGALYTAAVAAFVLYKCLQGKDETAVLHEEASKQQPLQSEQQLAQLTQQLAQTEQH

LNNLMAQLDPLFERVTTLAGAQQELLNMKLWTIHELLQDSKPDKDMEASEPGEGSGGE

SAGGGDKVSETGTFLISPHTEASRPLPEDFCLKEDEEEIGDSQAWEEPTNWSTETWNL

ATSWEVGRGLRRRCSQAVAKGPSHSLGWEGGTTAEGRLKQSLFS
.
.

....._ ..~...f..
SEQ ID NO: 69 v 539 by ..,...... ......~..,.
OVl7c, CCGGCCAATGCCGGACCGCTTCGGCACCGCCCGCCCGATCCCTCCACCCGTGGGCCGG

6159015-03 _CAATGGCGGGCGCAGTTTCGCTCTTGGGTGTGGTGGGGCTGCTGCTTGTGTCTGCGCT
DNA

equence GTCCGGGGTCCTAGGAGACCGCGCCAATCCCGACCTCCGGGCACACCCAGGGAACGCA

GAGGCAAGCAAGCAGCAGCCACTGCAGTCAGAGCAACAGCTGGCCCAGTTGACACAAC

AGCTGGCCCAGACAGAGCAGCACCTGAACAACCTGATGGCCCAGCTGGACCCCCTTTT

TGAGCGCCCAGCAGGAGCTTCTGAACATGAAGCTATGGACCATCCACGAGCTGCTGCA

AGATAGCAAGCCCGGAC

ORF Start: ATG at 61 ORF Stop: TAG at 526 SEQ ID NO: 70 155 as MW at 16521.5kD

OVl7c, MAGAVSLLGVVGLLLVSALSGVLGDRANPDLRAHPGNAAHPGSGATEPRRRPPLKDQR

rotein SequenceLAQTEQHLNNLMAQLDPLFERPAGASEHEAMDHPRAAAR

1$1 SEQ'.. LD.._~NO : 71 774 ~bp ~",. .

I~TOVI7d, GTGCGCGTTGGGGCGGCCGGCCAATGCCGGACCGCTTCGGCACCGCCCGCCCGATCCC

DNA

Sequence TGCTTGTGTCTGCGCTGTCCGGGGTCCTAGGAGACCGCGCCAATCCCGACCTCCGGGC

ACACCCAGGGAACGCAGCCCACCCCGGCTCTGGAGCCACGGAACCCCGGCGGCGACCA

CCGCTCAAGGATCAACGCGAGCGGACCCGGGCCGGGTCGCTGCCTCTGGGGGCGCTGT

ACACCGCGGCCGTCGCGGCTTTTGTGCTGTACAAGTGTTTGCAGGGGAAAGATGAAAC

TGCGGTTCTCCACGAGGAGGCAAGCAAGCAGCAGCCACTGCAGTCAGAGCAACAGCTG

GCCCAGTTGACACAACAGCTGGCCCAGACAGAGCAGCACCTGAACAACCTGATGGCCC

AGCTGGACCCCCTTTTTGAGCGGTGAGGAGAGCAATGATTCTGTGAATTTTTGGGGAA

TTTGTGGCAGGAGGGAGGAATGGGGACATAGGTTGGGAGCCACTGAGTGGACATTTCT

TCAGTGTGACTACTCTGGCTGGAGCCCAGCAGGAGCTTCTGAACATGAAGCTATGGAC

CATCCACGAGCTGCTGCAAGATAGCAAGCCGGACAAGGATATGGAGGCTTCAGAACCA

GGTGAAGGCTCGGGAGGCGAGTCTGCTGGAGGTGGAGACAAAGTCTCTGAAACTGGAA

CATTCCTGATCTCTCCCCCA

ORF Start: ATG at 77 ORF Stop: TGA at 488 _. .. .. ........ ...... ......
......................................................
', SEQ ID NO: 72 137 as MW at 14665.5kD

NOVl7d, MAGAVSLLGVVGLLLVSALSGVLGDRANPDLRAHPGNAAHPGSGATEPRRRPPLKDQR

Protein SequenceLAQTEQHLNNLMAQLDPLFER

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 17B.
Table 17B. Comparison of NOVl7a against NOVl7b through NOVl7d.
Protein Sequence NOVl7a Residues/ Identities/
Match Residues Similarities for the Matched Region NOVl7b 1..283 282/283 (99%) 52..334 283/283 (99%) NOVl7c 1..137 137/137 (100%) 1..137 137/137 (100%) NOVl7d 1..137 137/137 (100%)........
1..137 137/137 (100%) Further analysis of the NOV 17a protein yielded the following properties shown in Table 17C.
Table 17C. Protein Sequence Properties NOVl7a PSort 0.8200 probability located in outside; 0.1000 probability analysis: located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in lysosome (lumen) SignalP Cleavage site between residues 25 and 26 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 17D.
Table 17D. Geneseq Results for NOVl7a NOVl7a Identities/

Geneseq Protein/Organism/LengthResidues/ Similarities Expect Identifier[Patent #. Date] Match for MatchedValue the Residues Region ABB723.05Rat protein isolated 1..262 163/263(61%) 1e-79 from skin cells SEQ ID NO: 1..233 184/263(68%) Rattus sp, 242 aa.

[W0200190357-A1, 29-NOV-2001]

AAB88440Human membrane or secretory1..137 137/137(100%)2e-72 protein clone PSEC0222 1..137 137/137(100%) -Homo Sapiens, 139 aa.

[EP1067182-A2, 10-JAN-2001]

ABB68896Drosophila melanogaster85..224 33/140(23%) 0.001 polypeptide SEQ ID NO 816..943 54/140(38%) - Drosophila melanogaster, 2439 aa. [W0200171042-A2, 27-SEP-2001]

ABG28274Novel human diagnostic 136..269 34/140(24%) 0.47 protein #28265 - Homo 283..413 57/140(40%) Sapiens, 1121 aa.

[W0200175067-A2, ll-OCT-2001]

ABB64814Drosophila melanogaster59..172 29/120(24%) 0.81 polypeptide SEQ ID NO 2621..273154/120(44%) - Drosophila melanogaster, 3583 aa. [W0200171042-A2, 2~ SEP-2001] .. ...

In a BLAST search of public sequence datbases, the NOV 17a protein was found to have homology to the proteins shown in the BLASTP data in Table 17E.
Table 17E. Public BLASTP Results for NOVl7a NOVl7a Identities/

Protein Residues/ .Similarities Expedt AccessionProtein/Organism/Length Match for MatchedValue the Number Residues Portion Q8WV48 Similar to RIKEN cDNA 1..283 283/283(100%) e-163 1110032022 gene - Homo1..283 283/283(100%) Sapiens (Human), 283 aa.

Q9DCC3 1110032022Rik protein 1..262 153/262(58%) 1e-74 (Hypothetical 26.6 1..233 178/262(67%) kDa ~. . ... .... . ..
protein) - Mus musculus ... ... .. ... .
............ .. ..
.. .
.

(Mouse), 242 aa.

CAC39804 Sequence 247 from Patent1..137 137/137(100%) 5e-72 EP1067182 - Homo sapiens 1..137 137J137(100%) (Human), 139 aa.

Q9CTB6 1110032022Rik protein 35..262 133/228(58%) 4e-64 - Mus musculus (Mouse), 259 as 52..250 153/228(66%) (fragment).

Q9VMS2 CG14023 protein - Drosophila85..224 33/140 (23%) 0.004 melanogaster (Frui.t fly), 816..94354/140 (38%) 2439 aa.

PFam analysis predicts that the NOVl7a protein contains the domains shown in the Table 17F.
Table 17F. Domain Analysis of NOVl7a Identities/
Pfam Domain'NOVl7a Match Region Similarities Expect Value for the Matched Region No Significant Matches Found Example 18.
The NOVl8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 18A.

CATTGAGAAGCCAGAGAATCCTGAAACAACCCACACCTGGGACCCCCAGTGCATAAGC
ATGGAAGGCAAAATCCCCTATTTTCATGCTGGTGGATCCAAATGTTCAACATGGCCCT
TACCACAGCCCAGCCAGCACAACCCCAGATCCTCTTACCACAATATTACTGATGTGTG
TGAGCTGGCTGTGGGCCCTGCAGGTGCACCGGCCACTCTGTTGAATGAAGCAGGTAAA
GATGCTTTAAAATCCTCTCAAACCATTAAGTCTAGAGAAGAGGGAAAGGCAACCCAGC
AGAGGGAGGTAGAAAGCTTCCATTCTGAGACTGACCAGGATACGCCCTGGCTGCTGCC
CCAGGAGAAAACCCCCTTTGGCTCCGCTAAACCCTTGGATTATGTGGAGATTCACAAG
GTCAACAAAGATGGTGCATTATCATTGCTACCAAAACAGAGAGAGAACAGCGGCAAGC
CCAAGAAGCCCGGGACTCCTGAGAACAATAAGGAGTATGCCAAGGTGTCCGGGGTCAT
GGATAACAACATCCTGGTGTTGGTGCCAGATCCACATGCTAAAAACGTGGCTTGCTTT
GAAGAATCAGCCAAAGAGGCCCCACCATCACTTGAACAGAATCAAGCTGAGAAAGCCC
TGGCCAACTTCACTGCAACATCAAGCAAGTGCAGGCTCCAGCTGGGTGGTTTGGATTA
CCTGGATCCCGCATGTTTTACACACTCCTTTCACTGATAGCTTGACTAATGGAATGAT
TGGTTAAAATGTGATTTTTCTTCAGGTAACACTACAGAGTACGTGAAATGCTCAAGAA
TGTAGTCAGACTGACACTACTAAAGCTCCCAGCTCCTTTCATGCTCCATTTTTAACCA
CTTGCCTCTTTCTCCAGCAGCTGATTCCAGAACAAATCATTATGTTTCCTAACTGTGA
TTTGTAGATTTACTTTTTGCTGTTAGTTATAAAACTATGTGTTCAATGAAATAAAAGC
ACACTGCTTAGTATTCTTGAGGGACAATGCCAATAGGTATATCCTCTGGAAAAGGCTT
TCATGATTTGGCATGGGACAGACGGAAATGAAATTGTCAAAATTGTTTACCATAGAAA
GATGACAAAAGAAAATTTTCCACATAGGAAAATGCCATGAAAATTGCTTTTGAAAAAC
AACTGCATAACCTTTACACTCCTCGTCCATTTTATTAGGATTACCCAAATATAACCAT
TTAAAGAAAGAATGCATTCCAGAACAAATTGTTTACATAAGTTCCTATACCTTACTGA
CACATTGCTGATATGCAAGTAAGAAAT
ORF Start: ATG at 100~~~~, ~~ ORF Stop: TGA at 1891 ypro~nwwuw~°,~"SC'nYG'l:L~"'..~1:'.'m °.,:lY:lumv~,m ~.;,~wnmur~r, .n,'>~w'.ll.
SEQ ID NO: 74 ,597 as MW at 66638.8kD
18a, MKENVASATVFTLLLFLNTCLLNGQLPPGKPEIFKCRSPNKETFTCWWRPGTDGGLPT

tein Sequence DRKPYLWIKWSPPTLIDLKTGWFTLLYEIRLKPEKAAEWEIHFAGQQTEFKILSLHPG
QKYLVQVRCKPDHGYWSAWSPATFIQIPSDFTMNDTTVWISVAVLSAVICLIIVWAVA
LKGYSMVTCIFPPVPGPKIKGFDAHLLEKGKSEELLSALGCQDFPPTSDYEDLLVEYL
EVDDSEDQHLMSVHSKEHPSQGMKPTYLDPDTDSGRGSCDSPSLLSEKCEEPQANPST
FYDPEVIEKPENPETTHTWDPQCISMEGKIPYFHAGGSKCSTWPLPQPSQHNPRSSYH
NITDVCELAVGPAGAPATLLNEAGKDALKSSQTIKSREEGKATQQREVESFHSETDQD
TPWLLPQEKTPFGSAKPLDYVEIHKVNKDGALSLLPKQRENSGKPKKPGTPENNKEYA
KVSGVMDNNILVLVPDPHAKNVACFEESAKEAPPSLEQNQAEKALANFTATSSKCRLQ
LGGLDYLDPACFTHSFH
Further analysis of the NOVlBa protein yielded the following properties shown in Table 18B.
Table 18B. Protein Sequence Properties NOVl8a PSort 0.4600 probability located in plasma membrane; 0.1447 analysis: probability located in microbody (peroxisome); 0.1000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen) Si.gnalP Cleavage site between residues 25 and 26 analysis:

A search of the NOVl8a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 18C.

Table 18C. Geneseq Results for NOVl8a NOVlBa Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect Identifier[Patent #. Date] Match for Value the Residues MatchedRegion AAU99354 Human prolactin receptor1..597 597/622(95%)0.0 (PRLR) protein - Homo 1..622 597/622(95%) Sapiens, 622 aa.

[W0200250098-A2, 27-JUN-2002]

AAR10795 Human prolactin receptor1..597 597/622(95%)0.0 -Homo Sapiens, 622 aa. 1..622 597/622(95%) [US4992378-A, 12-FEB-1991]

AF1U99355 Human prolactin receptor1..597 596/622(95%)0.0 (PRLR) variant protein 1..622 597/622(95%) -Homo sapiens, 622 aa.

[W0200250098-A2, 27-JUN-2002]
~.

AAY95527 Human prolactin receptor1..311 311/336(92%)0.0 novel isoform - Homo 1..336 311/336(92%) Sapiens, 349 aa. [US6083753-A, 04-JUL-2000]

AAY96921 Soluble human prolactin1..311 311/336(92%)0.0 receptor clone F - Homo1..336 311/336(92%) Sapiens, 349 aa. [US6083714-A, 04-JUL-2000]

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

Table 18D. Public BLASTPResults for NOVl8a '~ ~ NOVlBa Identities/

Protein Residues/Similarities Expect AccessionProtein/Organism/Length Match for MatchedValue the Number Residues Portion P16471 Prolactin receptor precursor1..597 597/622(95%) 0.0 (PRL-R) - Homo Sapiens 1..622 597/622(95%) (Human), 622 aa.

Q9NOJ7 Prolactin receptor precursor1..597 531/622(85%) 0.0 Callithrix jacchus (Common1..622 555/622(88%) marmoset), 622 aa.

P14787 Prolactin receptor precursor1..597 450/624(72%) 0.0 (PRL-R) - Oryctolagus 1..616 496/624 (79%) cuniculus (Rabbit), 616 aa.

Q9XS92 Prolactin receptor precursor1..597 407/625 (65%) 0.0 - Trichosurus vulpecula 1..625 476/625 (76%) (Brush-tailed possum), 625 as.

A36116 prolactin receptor 2 7..597 406/618 (65%) 0.0 precursor - rat, 610 aa. 3..610 472/618 (75%) PFam analysis predicts that the NOV 18a protein contains the domains shown in the Table 18E.
"~,.,.,.:_.~w.
~,..,."~.:..j"~.~................._..._................................_Anal :.....................~...........,.........................~.............."...
................................_ Table 18E. Domain ysis of NOVl8a Identities/
Pfam Domain NOVl8a Match Region Similarities Expect Value for the Matched Region fn3 102..194 23/94 (24%) 0.051 58/94 (62%) Example 19.
The NOV 19 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 19A.
Table 19A. NOV19 Sequence Analysis SEQ ID N0: 75 2221 by AGCGGGCCGGGCGGCGGCGGGGAGATGCGGCTGCTGGCACTGGCGGCGGCCGCGCTGC
TGGCGCGGGCTCCGGCTCCGGAGGTCTGTGCGGCCCTCAATGTCACTGTGTCCCCGGG
GCCCGTGGTTGACTACCTGGAGGGGGAGAATGCCACTCTCCTCTGCCACGTCTCCCAG
AAAAGGCGGAAGGACAGCTTGCTGGCCGTGCGCTGGTTCTTTGCACACTCCTTCGACT
CCCAGGAGGCCTTGATGGTGAAGATGACCAAGCTCCGGGTGGTGCAGTACTATGGGAA
TTTCAGCCGCAGCGCCAAACGGCGGAGGCTGCGCCTGCTGGAGGAGCAGCGGGGGGCG
CTCTACAGGCTCTCCGTCTTGACACTGCAGCCCTCCGATCAAGGGCATTACGTCTGCA
GAGTCCAGGAAATCAGCAGGCACAGGAACAAGTGGACGGCCTGGTCCAATGGCTCCTC
AGCCACGGAAATGAGAGTCATTTCCCTCAAAGCTTCTGAAGAGTCATCCTTTGAGAAA
ACAAAAGAGACTTGGGCATTTTTTGAAGATCTCTATGTGTATGCTGTCCTCGTGTGCT
GCATGGGGATCCTCAGCATTCTGCTCTTCATGCTGGTCATCGTCTGGCAGTCTGTGTT
TAACAAGCGGAAATCCAGAGTGAGACATTATTTGGTGAAATGCCCTCAGAACAGCTCA
GGGGAGAGCTGTCACTAGCGTGACCAGCTTGGCCCCACTACAGCCCAAGAAGGGCAAG
AGGCAGAAGGAGAAGCCTGACATTCCTCCCGCAGTCCCTGCCAAAGCTCCGATAGCCC
CCACGTTCCATAAACCGAAGCTGCTGAAACCACAGAGAAAAGTCACGCTGCCAAAGAT
TGCTGAGGAAAACTTAACCTATGCCGAGCTGGAGCTGATCAAACCCCACCGGGCTGGC
AAAGGCGCCCCCACCAGCACTGTCTACGCCCAGATCCTCTTCGAGGAGAACAAGCTGT
AGTACAGCGTCCACCTCCAGGTTCTATTTAATACCTGCCACCCAGTGATTTATGAAGC
CTTGGAGACAAAGCCCTTATGTCTGTATTTTCACTCATGCCTTCTGAGTGGTGGGGAG
CCCCTTTTCAGCAGCATTCTGGGTGCCTTTGAAGAGGTACAAGCCTGCTCTCCCCAAA
AGAATCAGGGCCACAGCTCTTGACAGATCTCCCGGGACAAGATGCGCCTGGGTTTGAG
CCCTGAGCGTAAGGATTCTGATCCTGAGAGCAGCCAAGGAGATTTTCTGCTGAGCCAA
ACCCCTTCACATTTTTCTCCTCTTTCCCCAGGTTTTCTTTAAAATCGTTTTTAAATCT

TAATTTTACTCTCTACTCTTCCTGTATCCACGATACAAGCTCACAGTATATAGCTAGA
GGAAATGCCATTATGGACCCAACTGTAAGATGGCACATATGTTGGTTTTCCAAGGATC
AGATGGCATTGCAGGGCCACAGCCAACTGCTGATTGCCAGCACCACCTGAGATGGCAT
CTCTTGTTTTAAATAGATGCACTAACCCTGAAGATTAAGGCCAGAGGGGCAGACTGAC
TAGAGAAGTATAAGGTCTGTCTCTGAATGCCATGGTGCCCACCTATGAGACCCTGAGG
CCGCAGACAAAGAAGAACACCATTCTAGAGGGCTTCCAGCCCTTTCACAAGGTGGACC
TGTACTGATAGAGAAACACACTCTCTAAGAAGTGCTTACTCACCCTTTTCCAAAGGAG
CACAGGTGTTGGCCATCAGAAGACACACTGGAGCGCATGGGCCTCTTCACTGTGTGCC
AAGCTCAGTCACCTCTGATTCAGCCCCTGAGGGTGTCTGCTGCCAGGTGCCCTCAGGG
TAGGAGAGTGGGAAGTACACGCCAAGCTGGAAAGTGTGTTCTGAAGACCCTCCTCTTG
CCAAGTGCCTTGCCCATTGCAACCTTGTGTGTGAATTCTAATGGGTTTGAATGGGGGT
CAGGGTGCATGGGGAAGTTGCTCTGTGGACCTTTGGGACACAGGAATCTTGGACTTAC
TGGCAGGGGATCCATTCTGAAAGCACCATCCTGTCAACTGTGTTATTGAGGACATTTC
TTGATGTGAGTATAGTCTGGGTGGCTATTTACTGCCCACTATAGAAATTGTTTGACTA
TGTAGTGGACCATGTATATATGATAAATTATCTATTTTAAACAC
AAAAAAAGGGCGGCCGC
ORF Start: ATG at 25 ORF Stop: TAG at 712 SEQ ID NO: 76 229 as eMW at 26166.1kD
MRLLALAAAALLARAPAPEVCAALNVTVSPGPVVDYLEGENATLLCHVSQKRRKDSLL
AVRWFFAHSFDSQEALMVKMTKLRWQYYGNFSRSAKRRRLRLLEEQRGALYRLSVLT
LQPSDQGHYVCRVQEISRHRNKWTAWSNGSSATEMRVISLKASEESSFEKTKETWAFF
EDLYVYAVLVCCMGILSILLFMLVIVWQSVFNKRKSRVRHYLVI~CPQNSSGESCH
Further analysis of the NOV 19a protein yielded the following properties shown in Table 19B.
Table 19B. Protein Sequence Properties NOVl9a PSort 0.4600 probability located in plasma membrane; 0.2000 analysis: probability located in lysosome (membrane); 0.1000 probability located in endoplasmic retioulum (membrane); 0.1000 probability located in endoplasmic reticulum (lumen) SignalP Cleavage site between residues 23 and 24 analysis:
A search of the NOVl9a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 19C.
Table 19C. Geneseq Results for NOVl9a NOVl9a Identities/

Geneseq Protein/Organism/LengthResidues/:SimilaritiesExpect Identifier[Patent #~ Data] Match for the MatchedValue ResiduesRegion AAU18012 Human immunoglobulin 52..229 178/178 (100%)e-100 .

polypeptide SEQ ID No 17..194 178/178 (100%) Homo sapiens, 194 aa.

[W0200155315-A2, 02-AUG-2001]

AAU18070 Human immunoglobulin 14..190 174/177 (98%)2e-97 polypeptide SEQ IDINo 215 6..182 174/177 (98%) Homo Sapiens, 203 aa.

[W0200155315-A2, 02-AUG-2001]

ABB10520 Human cDNA SEQ ID NO: 14..190174/177 (98%) 2e-97 Homo Sapiens, 203 aa. 6..182 174/177 (98%) [W0200154474-A2, 02-AUG-2001]

ABB03217 Human musculoskeletal 14..190174/177 (98%) 2e-97 system related polypeptide SEQ ID 6..182 174/177 (98%) NO 1164 - Homo Sapiens, 203 aa. [W0200155367-A1, 02-AUG-2001]

ABB72358 Murine protein isolated 1..207 170/207 (82%) 1e-9'2 from skin cells SEQ ID NO: 682 - 3..206 185/207 (89%) Mus sp, 210 aa.

[W0200190357-A1, 29-NOV-2001]

In a BLAST search of public sequence datbases, the NOVl9a protein was found to have homology to the proteins shown in the BLASTP data in Table 19D.
Table 19D. Public BLASTP Results for NOVl9a NOVl9a Identities/

Protein Residues/Similarities Expect AccessionProtein/Organism/Length Match for the MatchedValue Number Residues Portion Q96MX7 CDNA FLJ31737 fis, clone1..164 155/164 (94%)1e-83 NT2RI2007084 - Homo 1..164 157/164 (95%) Sapiens (Human), 191 aa.

Q93033 Leukocyte surface protein38..226 47/189 (24%) 3e-04 -Homo sapiens (Human), 426..602 82/189 (42%) aa.

AAC72013 IG-LIKE MEMBRANE PROTEIN37..131 27/95 (28%) 4e-04 -Homo Sapiens (Human), 712..806 42/95 (43%) aa.

075054 KIAA0466 protein - Homo37..131 27/95 (28%) 4e-04 Sapiens (Human), 1214 712..806 42/95 (43%) as ( f ragment ) .

239207 leukocyte surface protein38..226 47/189 (24%) 0.002 V7 - human, 1021 aa. 426..602 81/189 (41%) PFam analysis predicts that the NOVl9a protein contains the domains shown in the Table 19E.
Table 19E. Domain Analysis of NOVl9a Pfam DomainjNOVl9a Match Region~Identities/ Expect Value Similarities for the Matched Regioxi ix 39..129 16/92 (17~) 2.7e-05 57/92 (62~) Example 20.
The NOV20 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 20A.
Table 20A. NOV20 Sequence Ai SEQ..LD.....NO.......77 ......... ... ................",.704 by L~OV20a, Sequence Start: ATG at 1 ~ORF Stop: TAG at 697 SEQ ID NO: 78 232 as MW at 24185.8kD
OV20a, MGDWSFLGRLLENAQEHSTVIGKVWLTVLFIFRILVLGAAAEDVWGDEQSDFTCNTRP

rotein Sequence HLLMTEQNWANQAAERQPPALKAYPAASTPAAPSPVGSSSPPLAHEAEAGAAPLLLDG
SGSSLEGSALAGTPEEEEQAVTTAAQMHQPPLPLGDPGRASKASRASSGRARPEDLAI
SEQ ID NO: 79 X1308 by 20b, CCGGGCTGCGAGAACGTCTGCTACGACAGGGCCTTCCCCATC
GGGCGCTGCAGATCATCTTCGTGTCCACGCCCACCCTCATCT
CAAGACGCTGTTCGAGGTGGGCTTCATCGCCGGCCAGTACTTTCTGT
CTGAAGCCGCTCTACCGCTGCGACCGCTGGCCCTGCCCCAACACGGT

Start: ATG at 1 ~ ~ORF Stop: TAG at 1306 SEQ ID N0: 80 X435 as BMW at 47427.5kD
NOV20b, MGDWSFLGRLLENAQEHSTVIGKVWLTVLFIFRILVLGAAAEDVWGDEQSDFTC

Protein Sequence RESPSPKEPPQDNPSSRDDRGRVRMAGALLRTYVFNIIFKTLFEVGFIAGQYFL
LKPLYRCDRWPCPNTVDCFISRPTEKTIFIIFMLAVACASLLLNMLEIYHLGWK
GVTSRLGPDASEAPLGTADPPPLPPSSRPPAVAIGFPPYYAHTAAPLGQARAVG
PPPAADFKMLALTEARGKGQSAKLYNGHHHLLMTEQNWANQAAERQPPALKAYP
PAAPSPVGSSSPPLAHEAEAGAAPLLLDGSGSSLEGSALAGTPEEEEQAVTTAA
ID NO: 81 954 by OV20c, equence CGTGTCCACGCCCACCCTCATCT
CGCCGGCCAGTACTTTCTGTACGGCTT
TCTTCATCATCTTCATGCTGGCGGTGGCCTGCGC
CCCCGAGGAGGAGGAGCAGGCCGTGACCACCGCGGCCCAGATGCACCAGCCGCCC
CCAGACCGGAGGACTTGGCCATCTAG
ORF Start: ATG at 1 ORF Stop: TAG at 952 SEQ ID N0: 82 X317 as BMW at 35397.1kD
20c, MGDWSFLGRLLENAQEHSTVIGKVWLTVLFIFRILVLGAAAEDVWGDEQSDFTC

tein Sequence ~RESPSPKEPPQDNPSSRDDRGRVRMAGALLRTYVFNIIFKTLFEVGFTAGQYFL
LKPLYRCDRWPCPNTVDCFISRPTEKTIFIIFMLAVACASLLLNMLEIYHLGWK
SGRARPEDLAI
Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table ZOB.
Table 20B. Comparison of NOV20a against NOV20b and NOV20c.
NOV20a Residues/-Identities/
Protein Sequence Match Residues Similarities for the Matched Region NOV20b 55..232 176/178 (98%) 258. .435,.... . 178/178 (99%) .... ..

NOV20c 147..232 69/86 (80%) 242..317 74/86 (85%) Further analysis of the NOV20a protein yielded the following properties shown in Table 20C.
Table 20C. Protein Sequence Properties NOV20a PSort 0.7900 probability located in plasma membrane; 0.3748 analysis: probability located in microbody (peroxisome); 0.3000 probability located in Golgi body; 0.2000 probability located in endoplasmic reticulum (membrane) SignalP Cleavage site between residues 42 and 43 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 NOY20a NOV20a Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect Identifier[Patent #. Data] Match for the Value ResiduesMatched Region AAW49009 Mouse alpha 3 connexin 58..232 88/175 (50%) 2e-38 protein - Mus sp, 417 267..417109/175 (62%) aa.

[W09830677-A1, 16-JUL-1998]

AAW23968 Connexin protein Cx40 1..59 43/59 (72%) 4e-20 - Homo sapiens, 358 aa. [WO9802150-1..59 48/59 (80%) A1, 22-JAN-1998]

AAG00107 Human secreted protein, 1..59 43/59 (72%) 7e-20 SEQ

ID N0: 4188 - Homo Sapiens,1..59 48/59 (80%) 83 aa. [EP1033401-A2, 2000]

AAB58122 Lung cancer associated 1..59 43/59 (72%) 7e-20 polypeptide sequence 48..106 48/59 (80%) SEQ ID

460 - Homo Sapiens, 124 aa.

[W0200055180-A2, 21-SEP-2000]

ABB05038 Human NOV3b protein SEQ 1..59 40/59 (67%) 4e-l9 ID

N0:12 - Homo Sapiens, 1..59 47/59 (78%) 543 aa.

[W0200190155-A2, 29-NOV-2001]

In a BLAST search of public sequence datbases, the NOV20a 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 Tdentities/

Residues/'Similarities Expect AccessionProtein/Organism/Length Match for the MatchedValue Number ResiduesPortion Q9Y6H8 Gap junction alpha-3 55..232 176/178 (98%) 8e-99 protein (Connexin 46) (Cx46) 257..434178/178 (99%) - Homo Sapiens (Human), 434 aa.

~.~,.~..w.:~~r Q64448 Gap junction alpha-3 58..232 88/175 (50%) 6e-38 protein (Connexin 46) (Cx46) 266..416109/175 (62%) - Mus musculus (Mouse), 416 aa.

525764 connexin 46 - rat, 416 55..232 90/178 (50%) 2e-35 aa.

264..416107/178 (59%) P29414 Gap junction alpha-3 55..232 90/178 (50%) 2e-35 protein (Connexin 46) (Cx46) 263..415107/178 (59%) -Rattus norvegicus (Rat), aa.

A45338 connexin-56 - chicken, 1..59 56/59 (94%) 1e-26 aa. 1..59 X58/59 (97%) PFam analysis predicts that the NOV20a protein contains the domains shown in the Table 20F.
Table 20F. Domain Analysis of NOV20a identities/
Pfam DomainNOV20a Match Region Similarities Expect Value for the Matched Region connexin 1..118 65/247 (26%) 1.4e-09 89/247 (36%) Example 21.
The NOV21 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 21A.

ATCTATAGGACCGCCTACCGCCGCAGCCCTGGGCTGGCCCCTGCCAGGCCTC
CGTGCTGCCCCGGCTGGAAGAGGACCAGCGGGCTTCCTGGGGCCTGTGGAGC
ATGCCAGCCGCCATGCCGGAACGGAGGGAGCTGTGTCCAGCCTGGCCGCTGC
CTGGTGCACTCCTTCCAGCAGCTCGGCCGCAT
CTCCTCTTCCTCCTCCCCTTCCTCGGGAGGCTCCCCAGACCCTGGCATGGGATGGGCT
GGGATCTTCTCTGTGAATCCACCCCTGGCTACCCCCACCCTGGCTACCCCAACGGCAT
GACCCCCAGCACAATAAAAATGAAAC
Start: ATG at 96 ~ORF Stop: TGA at 915 SEQ ID N0: 84 X273 as BMW at 29617.4kD
~TOV2la, MRGSQEVLLMWLLVLAVGGTEHAYRPGRRVCAVRAHGDPVSESFVQRVYQPFLTTCDG

Protein Sequence CVQPGRCRCPAGWRGDTCQSDVDECSARRGGCPQRCVNTAGSYWCQCWEGHSLSADGT
LCVPICGGPPRVAPNPTGVDSAMKEEVQRLQSRVDLLEEKLQLVLAPLHSLASQALEHG
~LPDPGSLLVHSFQQLGRIDSLSEQISFLEEQLGSCSCKKDS
SEQ ID NO: 85 1307 by ~10V21b, CCAAGCTGGCCCTGCACGGCTGCAAGGGAGGCTCCTGTGGACAGGCCAGG

Sequence ~CATCTCCAGTCCCAGGACACAGCAGCGGCCACCATGGCCACGCCTGGGCT
CGTTCGTGCAGCGTGTGTAC
ACACTCTGTGTGCC
CCCGGACCCCGGCAGCCTCCTGGTGCACTCCTTC
Start: ATG at 150 ~ ~ORF Stop: TGA at 897 SEQ ID NO: 86 249 as MW at 25902.OkD
21b,-~ MATPGLQQHQQPPGPGRHRWPPPPGGAAPAPVRGMTDSPPPAVGCVLSGLTGTLSP
2113-06 ~SCSVCTSPSSPPATGTGPAAPTAICQPPCRNGGSCVQPGRCRCPAGWRGDTCQSDV

ein Sequence VQRLQSRVDLLEEKLQLVLAPLHSLASQALEHGLPDPGSLL' SFLEEQLGSCSCKKDS
~1".F.f'~y ID '~NO: y87 ....._ ... .~ ° 841 by L~10V21C, _CACCCiGATCCACCA'1'C;ACiCiCiCiC:'1'C:'1'(:ACiciAhlz'tuiv:wm:wuit-~-lu3WUitW:l iu:wv~umm Sequence CTCACGGGGACCCTGTCTCCGAGTCGTTCGTGCAGCGTGTGTACCAGCCCTTCCTCAC
CACCTGCGACGGGCACCGGGCCTGCAGCACCTACCGAACCATCTATAGGACCGCCTAC
hrrrCGr_ArCC_CTGGCCTGGCCCCTGCCAGGCCTCGCTACGCGTGCTGCCCCGGCTGGA
TGTGGATGAATGCAGTGCTAGGAGGGGCGGCTGTCCCCAGC
ORF Start: at 2 ~RF Stop: end of sequence SEQ ID NO: 88 280 as MW at 30235.OkD
OV2lc, TGSTMRGSQEVLLMWLLVLAVGGTEHAYRPGRRVCAVR.AHGDPVSESFVQRVYQPF

rotein Sequence NGGSCVQPGRCRCPAGWRGDTCQSDVDECSARRGGCPQRCVNTAGSYWCQCWEGH~
ADGTLCVPKGGPPRVAPNPTGVDSAMKEEVQRLQSRVDLLEEKLQLVLAPLHSLA~
LEHGLPDPGSLLVHSFQQLGRIDSLSEQISFLEEQLGSCSCKKDSVDG
SEQ.......ID...N~...~....._$..9............. ....................
~........................................................... 769.......~p.
OV2ld, _CACCGGATCCTACCGGCCCGGCCGTAGGGTGTGTGCTGTCCGGGCTCACGGGGACCCT

equence ACCGGGCCTGCAGCACCTACCGAACCATCTATAGGACCGCCTACCGCCGCAGCCCTGG
GCTGGCCCCTGCCAGGCCTCGCTACGCGTGCTGCCCCGGCTGGAAGAGGACCAGCGGG
rTTCCT~c~GGCCTGTGGAGCAGCAATATGCCAGCCGCCATGCCGGAACGGAGGGAGCT
ACAC
TGCAGCTGGTGCTGGCCCCACTGCACAGCCTGGCCTCGCAGGCACTGGAGCATGGGC
CCCGGACCCCGGCAGCCTCCTGGTGCACTCCTTCCAGCAGCTCGGCCGCATCGACTC
CTGAGCGAGCAGATTTCCTTCCTGGAGGAGCAGCTGGGGTCCTGCTCCTGCAAGAAA
ORF Start: at 2 ORF Stop: end of sequence SEQ ID N0: 90 256 as MW at 27640.9kD
NOV2ld, TGSYRPGRRVCAVRAHGDPVSESFVQRVYQPFLTTCDGHRACSTYRTIYRTAS

Protein Sequence DVDECSARRGGCPQRCVNTAGSYWCQCWEGHSLSADGTLCVPKGGPPRVAPNF
AMKEEVQRLQSRVDLLEEKLQLVLAPLHSLASQALEHGLPDPGSLLVHSFQQI
LSEQISFLEEQLGSCSCKKDSVDG
SEQ ID NO: 91 841 by 1e CAC:~'1'c~~GACGGGCACCGGGCCTGCAGCACCTACCGAACCATCTATAGGACCGCCTAC
CGCCGCAGCCCTGGGCTGGCCCCTGCCAGGCCTCGCTACGCGTGCTGCCCCGGCTGGA
AGAGGACCAGCGGGCTTCCTGGGGCCTGTGGAGCAGCAATATGCCAGCCGCCATGCCG
GAACGGAGGGAGCTGTGTCCAGCCTGGCCGCTGCCGCTGCCCTGCAGGATGGCGGGGT
GACACTTGCCAGTCAGATGTGGATGAATGCAGTGCTAGGAGGGGCGGCTGTCCCCAGC
' GCTGCGTCAACACCGCCGGCAGTTACTGGTGCCAGTGTTGGGAGGGGCACAGCCTGTC
TGCAGACGGTACACTCTGTGTGCCCAAGGGAGGGCCCCCCAGGGTGGCCCCCAACCCG
CCCGGACCCCGGCAGCCTCCTGGTGCACTCCTTCCAGCAGCTC
ORF Start: at 2 p'v' ORF Stop: end of sequence ~"~,_.~., SEQ ID N0: 92 280 as "'MW at 30235.OkD~n~
OV2le, TGSTMRGSQEVLLMWLLVLAVGGTEHAYRPGRRVCAVRAHGDPVSESFVQRVY

rotein Sequence NGGSCVQPGRCRCPAGWRGDTCQSDVDECSARRGGCPORCVNTAGSYWCOCWE
LEHGLPDPGSLLVHSFQQLGRIDSLSEQISFLEEQLGSCSCKKDSVDG
SEQ ID NO: 93 769 by OV2lf, CACCGGATCCTACCGGCCCGGCCGTAGGGTGTGTGCTGTCCGGGCTCACGGGGACCCT

equence ACCGGGCCTGCAGCACCTACCGAACCATCTATAGGACCGCCTACCGCCGCAGCCCTGG
GCTGGCCCCTGCCAGGCCTCGCTACGCGTGCTGCCCCGGCTGGAAGAGGACCAGCGGG
CTTCCTGGGGCCTGTGGAGCAGCAATATGCCAGCCGCCATGCCGGAACGGAGGGAGCT
GTGTCCAGCCTGGCCGCTGCCGCTGCCCTGCAGGATGGCGGGGTGACACTTGCCAGTC
AGATGTGGATGAATGCAGTGCTAGGAGGGGCGGCTGTCCCCAGCGCTGCGTCAACACC
~ruwrGUACrccY°rCCAGCAGCTCGGCCGCATCGACTC
CCTGGAGGAGCAGCTGGGGTCCTGCTCCTGCAAGAAA
ORF Start: at 2 H~ORF Stop: end of ~~.~"~.~,~,~ _ _~,~ __ . _ _....__. r-. .._ _ _....___. .... . _ .. ~..
sequence,-.~ _._.
SEQ ID NO: 94 256 as MW at 27640.9kD
OV2lf TGSYRPGRRVCAVRAHGDPVSESFVQRVYQPFLTTCDGHRACSTYRTIYRTAYRRSPG

rotein Sequence DVDECSARRGGCPQRCVNTAGSYWCQCWEGHSLSADGTLCVPKGGPPRVAPNPTGVDS
AMKEEVQRLQSRVDLLEEKLQLVLAPLHSLASQALEHGLPDPGSLLVHSFQQLGRIDS
LSEQISFLEEQLGSCSCKKDSVDG
SEQ ID NO: 95 1475 by 21g, CTGGTGTTGGCAGTGGGCGGCACAGAGCACGCCTACCGGCCCGGCCGT

TATAGGACCGCCTACCGCCGCAGCCCTGGGCTGGCCCCTGCCAGGCCTCGCTACGCGT

GCTGCCCCGGCTGGAAGAGGACCAGCGGGCTTCCTGGGGCCTGTGGAGCAGCAATATG

CCAGCCGCCATGCCGGAACGGAGGGAGCTGTGTCCAGCCTGGCCGCTGCCGCTGCCCT

GCAGGATGGCGGGGTGACACTTGCCAGTCAGATGTGGATGAATGCAGTGCTAGGAGGG

GCGGCTGTCCCCAGCGCTGCGTCAACACCGCCGGCAGTTACTGGTGCCAGTGTTGGGA

GGGGCACAGCCTGTCTGCAGACGGTACACTCTGTGTGCCCAAGGGAGGGCCCCCCAGG

GTGGCCCCCAACCCGACAGGAGTGGACAGTGCAATGAAGGAAGAAGTGCAGAGGCTGC

AGTCCAGGGTGGACCTGCTGGAGGAGAAGCTGCAGCTGGTGCTGGCCCCACTGCACAG

CCTGGCCTCGCAGGCACTGGAGCATGGGCTCCCGGACCCCGGCAGCCTCCTGGTGCAC

TCCTTCCAGCAGCTCGGCCGCATCGACTCCCTGAGCGAGCAGATTTCCTTCCTGGAGG

AGCAGCTGGGGTCCTGCTCCTGCAAGAAAGACTCGTGACTGCCCAGCGCCCCAAGCTG

GACTGAGCCCCTCACGCCGCCCTGCAGCCCCCATGCCCCTGCCCAACATGCTGGGGGT

CCAGAAGCCACCTCGGGGTGACTGAGCGGAAGGCCAGGCAGGGCCTTCCTCCTCTTCC

TCCTCCCCTTCCTCGGGAGGCTCCCCAGACCCTGGCATGGGATGGGCTGGGATCTTCT

CTGTGAATCCACCCCTGGCTACCCCCACCCTGGCTACCCCAACGGCATCCCAAGGCCA

GGTGGGCCCTCAGCTGAGGGAAGGTACGAGCTCCCTGCTGGAGCCTGGGACCCATGGC

ACAGGCCAGGCAGCCCGGAGGCTGGGTGGGGCCTCAGTGGGGGCTGCTGCCTGACCCC

CAGCACAATAAAAATGAAACGTGAC
_..................................... .....................
......

p-ORF Start. at 201 ORF Sto . TGA at 1080 SEQ ID NO: 96 293 as MW at 31986.2kD
W.._" ,, 21g, LILLRQATQRRRPPRLEAQAMRGSQEVLLMWLLVLAVGGTEHAYRPGRRVCAVRAHGD

tein SequenceGLPGACGAAICQPPCRNGGSCVQPGRCRCPAGWRGDTCQSDVDECSARRGGCPQRCVN

TAGSYWCQCWEGHSLSADGTLCVPKGGPPRVAPNPTGVDSAMKEEVQRLQSRVDLLEE

KLQLVLAPLHSLASQALEHGLPDPGSLLVHSFQQLGRIDSLSEQISFLEEQLGSCSCK

KDS

..,n........gEQ ID NO: 97 1384 by . ' ...... .......................... .
..........................................................
... .. ... .......
1h, ................................ .
...... ,.~....~.._.,.., TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

DNA

uence TTTTTTGTCACGTTTCATTTTTATTGTGCTGGGGGTCAGGCAGCAGCCCCCACTGAGG

CCCCACCCAGCCTCCGGGCTGCCTGGCCTGTGCCATGGGTCCCAGGCTCCAGCAGGGA

GCTCGTACCTTCCCTCAGCTGAGGGCCCACCTGGCCTTGGGATGCCGTTGGGGTAGCC

AGGGTGGGGGTAGCCAGGGGTGGATTCACAGAGAAGATCCCAGCCCATCCCATGCCAG

GGTCTGGGGAGCCTCCCGAGGAAGGGGAGGAGGAAGAGGAGGAAGGCCCTGCCTGGCC

TTCCGCTCAGTCACCCCGAGGTGGCTTCTGGACCCCCAGCATGTTGGGCAGGGGCATG

GGGGCTGCAGGGCGGCGTGAGGGGCTCAGTCCAGCCTGGGGCGCTGGGCAGTCACGAG

TCTTTCTTGCAGGAGCAGGACCCCAGCTGCTCCTCCAGGAAGGAAATCTGCTCGCTCA

GGGAGTCGATGCGGCCGAGCTGCTGGAAGGAGTGCACCAGGAGGCTGCCGGGGTCCGG

GAGCCCATGCTCCAGTGCCTGCGAGGCCAGGCTGTGCAGTGGGGCCAGCACCAGCTGC

AGCTTCTCCTCCAGCAGGTCCACCCTGGACTGCAGCCTCTGCACTTCTTCCTTCATTG

CACTGTCCACTCCTGTCGGGTTGGGGGCCACCCTGGGGGGCCCTCCCTTGGGCACACA

GAGTGTACCGTCTGCAGACAGGCTGTGCCCCTCCCAACACTGGCACCAGTAACTGCCG

GCGGTGTTGACGCAGCGCTGGGGACAGCCGCCCCTCCTAGCACTGCATTCATCCACAT

CTGACTGGCAAGTGTCACCCCGCCATCCTGCAGGGCAGCGGCAGCGGCCAGGCTGGAC

ACAGCTCCCTCCGTTCCGGCATGGCGGCTGGCATATTGCTGCTCCACAGGCCCCAGGA

AGCCCGCTGGTCCTCTTCCAGCCGGGGCAGCACGCGTAGCGAGGCCTGGCAGGGGCCA

GCCCAGGGCTGCGGCGGTAGGCGGTCCTATAGATGGTTCGGTAGGTGCTGCAGGCCCG

GTGCCCGTCGCAGGTGGTGAGGAAGGGCTGGTACACACGCTGCACGAACGACTCGGAG

ACAGGGTCCCCGTGAGCCCGGACAGCACACACCCTACGGCCGGGCCGGTAGGCGTGCT

CTGTGCCGCCCACTGCCAACACCAGAAGCCACATCAGCAGCACCTCCTGAGAGCCCCT

CATGGCCTGTGCCTCCAGGCGGGGTGGCCTTCTCCTCTGGTTCTTGGGGA

ORF Start: ATG at 209 ORF Stop: TGA at 482 SEQ ID NO: 98 91 as MW at 9729.9kD

'21h, MGPRLQQGARTFPQLRAHLALGCRWGSQGGGSQGWIHREDPSPSHARVWGASRGRGGG

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 21B.

Table 21B. Comparison of NOV2la against NOV2lb through NOV2lj.

N~V2la Residues/
2dentities/

Protein Similarities Sequence for Match the Residues Matched Region NOV2lb79..273 176/196 (89%) 54..249 179/196 (90%) NOV21C1..273 273/273 (100%) 5..277 273/273 (100%) NOV2ld23..273 ~ 250/251 (99%) 3.,253 251/251 (99%) NOV2le1.._73. 273/273 (100%) ~~
5..277 273/273 (100%) NOV2lf23..273 250/251 (99%) 3..253 251/251 (99%) NOV2lg1..273 273/273 (100%) 21..293 273/273 (100%) NOV2lhNo Significant AlignmentFound.

NOV2li1..273 273/273 (100%) 1..273 273/273 (100%) NOV2lj1..273 ~.....__............ . (99%) 1..273 273/273 (99%) Further analysis of the NOV21 a protein yielded the following properties shown in Table 21C.
Table 21C. Protein Sequence Properties NOV2la PSort 0.5500 probability located, in endoplasmic reticulum analysis: (membrane); 0.1900 probability located in lysosome ilumen);
0.1000 probability located in endoplasmic reticulum (lumen);
0.1000 probability located in outside SignalP Cleavage site between residues 23 and 24 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 21D.

Table 21D. Geneseq Results for NOV2la NOV2la Identities/

Geneseq Protein/Organism/LengthResidues/Similarities Expect Identifier[Patent #~ Date] Match for MatchedValue the Residues Region AAB61609 Human protein HP03375 1..273 273/273(100%) e-168 - Homo Sapiens, 273 aa. 1..273 273/273(100%) [W0200102563-A2, 11-JAN-2001]

AAM23991 Human EST encoded protein1..273 273/273(100%) e-168 SEQ ID N0: 1516 - Homo 1..273 273/273(100%) Sapiens, 273 aa.

[W0200154477-A2, 02-AUG-2001]

AAB01376 Neuron-associated protein1..273 273/273(100%) e-168 -Homo Sapiens, 273 aa. 1..273 273/273(100%) [W0200034477-A2, 15-JUN-2000]

AAB24044 Human PR01449 protein 1..273 273/273(100%) e-168 'sequence SEQ ID N0:8 1..273 273/273(100%) - Homo Sapiens, 273 aa.

[W0200053754-A1, 14-SEP-2000]

AAB18675 Amino acid sequence 1..273 273/273(100%) e-168 of a human a PR01449 polypeptide1..273 273/273(100%) - Homo Sapiens, 273 aa.

[W0200053752-A2, 14-SEP-2000]

In a BLAST search of public sequence datbases, the NOV2la protein was found to have homology to the proteins shown in the BLASTP data in Table 21E.
Table 21E. Public BLASTPResults for NOV2la NOV2la Identities/

Protein Residues/;Similarities Expect AccessionProtein/Organism/LengthMatch for MatchedValue the Number Residues Portion Q9UHF1 NOTCH4-like protein 1..273 273/273(100%) e-168 (Hypothetical 29.6 kDa 1..273 273/273(100%) protein) - Homo Sapiens (Human), 273 aa.

Q96EG0 ,Similar to NEU1 protein1..273 272/273(99%) e-167 -Homo Sapiens (Human), 1..273 273/273(99%) aa.

CAC38966 Sequence 17 from Patent1..273 234/273 (85%) e-136 W00119856 - Homo Sapiens 1..234 234/273 (85%) (Human), 234 aa.

Q9QXT5 NOTCH4-like protein 1..272 214/274 (78%) e-129 (Vascular endothelial zinc 4..277 232/274 (84%) finger 1) - Mus musculus (Mouse), 278 aa.

Q9DCP5 Vascular endothelial 1..272 203/274 (74%) e-119 zinc finger 1 - Mus musculus 4..264 220/274 (80%) (Mouse), 265 aa.

PFam analysis predicts that the NOV2la protein contains the domains shown in the Table 21F.
Table 21F. Domain Analysis of NOV2la Identities/
Pfam DomainiNOV2la Match Region Similarities Expect Value for the Matched Region EGF 107..134 15/47 (32%) 0.0037 22/47 (47%) EGF 141..176 15/47 (32%) 0.0012 25/47 (53%) Example 22.
The NOV22 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 22A.

TGGACAGCCAGACTAGGTGGGGGCAG
RF Start: ATG at 31 ORF Stop: TAG at 1291 ID N0: 104 X420 as BMW at 45678.7kD
22a, MAMDAGNPPLNSTVPWIEVFDENDNPPTFSKPAYFVSWENIMAGA'1'VLFLNATDLD

tein Sequence KTGIATVNITLLDINDNHPTWKDAPYYINLVEMTPPDSDVTTWAVDPDLGENGTLVY
SIQPPNKFYSLNSTTGKIRTTHAMLDRENPDPHEAELMRKIWSWDCGRPPLKATSS
ATVFVNLLDLNDNDPTFQNLPFVAEVLEGIPAGVSIYQWAIDLDEGLNGLVSYRMPV
GMPRMDFLINSSSGVVVTTTELDRERIAEYQLRWASDAGTPTKSSTSTLTIHVLDVN
DETPTFFPAWNVSVSEDVPREFRVVWLNCTDNDVGLNAELSYFITGAAPASAHLCRP
ID NO: 105 1113 by OV22b, GGATCCGCCACAGACCTGGACCGCTCCCGGGAGTACGGCCAGGAGTCCATCATCTACT

equence GTCTCTGCTTGACCGAGAGACCAAGTCTGAATACATCCTCATCGTTCGCGCAGTGGAC
GGGGGTGTGGGCCACAACCAGAAAACTGGCATCGCCACCGTAAACATCACCCTCCTGG
ACATCAATGACAACCACCCCACGTGGAAGGACGCACCCTACTACATCAACCTGGTGGA
GATGACCCCTCCAGACTCTGATGTGACCACGGTGGTGGCTGTTGACCCAGACCTGGGA
CCACGCCATGCTGGACCGGGAGAACCCCGACCC
TACCAAGTGGTGGCCATCGACCTCGATGAGGGCCTGAACGGCCTG
TGCCGGTGGGCATGCCCCGCATGGACTTCCTCATCAGCAGCAGCA
TG
CACCCCTCCCAGATGGACAGC
~ORF Start: at 1 ORF Stop: end of sequence __ .' ~'~L.~~~.NU
SEQ ID N0: 106 371 as MW at 40369.7kD
L~OV22b, GSATDLDRSREYGQESIIYSLEGSTQFRINARSGEITTTSLLDRETKSEYILIVRAVD

Protein Sequence ENGTLWSIQPPNKFYSLNSTTGKIRTTHAMLDRENPDPHEAELMRKIWSWDCGRP
PLKATSSATVFVNLLDLNDNDPTFQNLPFVAEVLEGIPAGVSIYQWAIDLDEGLNGL
VSYRMPVGMPRMDFLISSSSGVWTTTELDRERIAEYQLRWASDAGTPTKSSTSTLT
IHVLDVNDETPTFFPAWNVSVSEDVPREFRVVWLNCTDNDVGLNAELSYFITGAAPA
SAHLCRPPGALPPPLPDGQPDLE
SEQ ID NO: 107 1114 by NOV22c, _GGATCCGCCACAGACCTGGACCGCTCCCGGGAGTACGGCCAGGAGTCCATCATCTACT

Sequence CGTCTCTGCTTGACCGAGAGACCAAGTCTGAATACATCCTCATCGTTCGCGCAGTGGA
~CGGGGGTGTGGGCCACAACCAGAAAACTGGCATCGCCACCGTAAACATCACCCTCCTG

CTACAGCCTCAAC
CTCTGAAAGCCACCAGCAGTGCCACAGTGTTTGTGAACCTCTTGGATCTCAATGAC
TGACCCCACCTTTCAGAACCTGCCTTTTGTGGCCGAGGTGCTTGAAGGCATCCCGG
GGGGTCTCCATCTACCAAGTGGTGGCCATCGACCTCGATGAGGGCCTGAACGGCCT
CGCGAAGTACCAGC
TCCATGTGCTGGATGTGAACGACGAGACGCCCACCTTCTTCCCGGCCGTGTACAAT
TGCAGAGCTCAGCTACTTCATCACAGGTGCTGCCCCGGC
ORF Start: at 2 ORF Stop: end of sequence ___.,....
SEQ ID N0: 108 371 as MW at 40080.6kD
NOV22c, DPPQTWTAPGSTARSPSSTPWKAPPSFGSMPAPGEITTTSLLDRETKSEYILIVRAVD

Protein Sequence ENGTLWSIQPPNKFYSLNSTTGKIRTTHAMLDRENPDPHEAELMRKIWSVTDCGRP
PLKATSSATVFVNLLDLNDNDPTFQNLPFVAEVLEGIPAGVSIYQWAIDLDEGLNGL
IHVLDVNDETPTFFPA
ID N0: 109 X1114 by L~TOV22d, _GGATCCGCCACAGACCTGGACCGCTCCCCGGGAGTACGGCCAGGAGTCCATCATCTAC

Sequence CGTCTCTGCTTGACCGAGAGACCAAGTCTGAATACATCCTCATCGTTCGCGCAGTGGA
CGGGGGTGTGGGCCACAACCAGAAAACTGGCATCGCCACCGTAAACATCACCCTCCTG
CCATGAGGCCGAGCTGATGCGCAAAATCGTCGTCTCTGTTACTGACTGTGGCAGGCC
CGGGGGTCTCCATCTACCAAGTGGTGGCCATCGACCTCGATGAGGGCCTGAACGGCCT
TCGCGGAGTACCAGC
AGAGCTCAGCTACTTCATCACAGGTGCTGCCCCGGC
CCTGGGGCCCTGCCTCCACCCCTCCCAGATGGACAG
ORF Start: at 2 ORF Stop: end of sequence ;:..
SEQ ID NO: 110 371 as MW at 40487.9kD
NOV22d, DPPQTWTAPREYGQESIIYSLEGSTQFRINARSGEITTTSLLDRETKSEYILIVRAV

Protein Sequence ENGTLVYSIQPPNKFYSLNSTTGKIRTTHAMLDRENPDPHEAELMRKIWSVTDCGR
PLKATSSATVFVNLLDLNDNDPTFQNLPFVAEVLEGIPAGVSIYQWAIDLDEGLNG
VSYRMPVGMPRMDFLINSSSGVWTTTELDRERIAEYQLRWASDAGTPTKSSTSTL
IHVLDVNDETPTFFPAVYNVSVSEDVPREFRVVWLNCTDNDVGLNAELSYFITGAAP
SAHLCRPPGALPPPLPDGQPDLE

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 22B.
Table 22B. Comparison of NOV22a against NOV22b through NOV22e.
Protein Sequence~NOV22a Residues/Ident~.ties/

Match ResiduesSsmilarities for the Matched Region NOV22b 53..420 366/368 (99%) 2..369 368/368 (99%) NOV22c 85..420 333/336 (99%) 34..369 334/336 (99%) NOV22d 61..420 360/360 (100%) 10..369 360/360 (100%) T_._ NOV22e 53..407 346/355 (97%) 2..352 347/355 (97%) Further analysis of the NOV22a protein yielded the following properties shown in Table 22C.
Table 22C. Protein Sequence Properties NOV22a PSort 0.7900 probability located in plasma membrane; 0.3000 analysis: probability located in microbody (peroxisome); 0.3000 probability located in Golgi body; 0.2000 probability located in endoplasmic reticulum (membrane) SignalP 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/Similarities Expect Identifier[Patent #~ Date] Match for MatchedZTalue the ResiduesRegion AAM39046 Human polypeptide NO 1..420 418/420(99%) 0.0 SEQ ID

2191 - Homo Sapiens,aa. 127..546419/420(99%) [W0200153312-A1, 2001]

AAM38969 Human polypeptide NO 1..420 418/420(99%) 0.0 SEQ ID

2114 - Homo Sapiens,aa. 139..558419/420(99%) [W0200153312-A1, 2001]

AAU01093 Gene 24 Human secreted 1..382 382/382(100%)0.0 protein homologous 68..449 382/382(100%) amino acid sequence - Homo Sapiens, 449 aa.

[W0200123402-A1, 2001]

ABG03875 Novel human diagnostic 85..395 306/402(76%) e-161 protein #3866 - Homo 994..1390306/402(76%) Sapiens, 1509 aa.

[W0200175067-A2, 2001]

AAM40755 Human polypeptide NO 123..395262/273(95%) e-148 SEQ ID

5686 - Homo Sapiens,aa. 6..278 263/273(95%) [W0200153312-A1, 2001]

In a BLAST search of public sequence datbases, the NOV22a protein was found to have homology to the proteins shown in the BLASTP data in Table 22E.
Table 22E. Public BLASTP Results for NOV22a Protein NOV22a Identities) Residues/Similarities Expect AccessionProtein/Organism/Length for Match the Value Number Matehed Residues Portion AAH32581 Similar to cadherin 1..420 420/420(100%) 0.0 related X

23 - Homo Sapiens (Human),642..1061420/420(100%) 1061 aa.

Q96JL3 KIAA1812 protein - Homo1..395 395/395(100%) 0.0 sapiens (Human), 803 233..627 395/395(100%) as (fragment).

Q9H251 Cadherin-23 precursor 1..395 395/395(100%) 0.0 (Otocadherin) - Homo 642..1036395/395(100%) Sapiens (Human), 3354 aa.

P58365 Cadherin 23 precursor 1..394 377/394(95%) 0.0 (Otocadherin) - Rattus 640..1033385/394(97%) norvegicus (Rat), 3317 aa.

Q99PF4 Cadherin 23 precursor 1..394 374/394(94%) 0.0 ~

(Otocadherin) - Mus 642..1035384/394(96%) musculus (Mouse), 3354 aa.

PFam analysis s the predicts domains that shown the NOV22a in protein the contain Table 22F.
Table 22F. Domain Analysis of NOV22a Identities) Pfam DomainNOV22a Match Region Similarities Expect Value for the Matched Region cadherin 35..128 41/108 (38%) ~ 6.2e-17 67/108 (62%) cadherin 142..238 36/112 (32%) 3.1e-11 67/112 (60%) cadherin 254..345 41/107 (38%) 1.9e-24 69/107 (64%) Example 23.
The NOV23 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 23A.
Table 23A. NOV23 Sequence Analysis SEQ ID NO: 113 X1772 by NOV23a, CTTTTGCACTGATCATTTCTCTTAATTGGCAGGTAACAA

Sequence AAGGGATCACTGTGCTGGGTTTAAATGCGGTATTTGACA
CAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGA
CAATGGGCTGCA
Ac:C:AAGAAAAAGAAACTCATTGCCGCTGTCGTGCTGGGAATCTTATTCATCAACACGC
TGAGATGTGTGCTGCGCAGCGGCGAGTGGCGGAGTGAGGAACAGCTTTTCAGAAGTGC
TCTGTCTGTGTGTCCCCTCAATGCTAAGGTACACTACAACATTGGCAAAAACCTGGCT
GATAAAGGCAACCAGACAGCTGCCATCAGATACTACCGGGAAGCTGTAAGATTAAATC
CCAAGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAA.GAAAGGAATGAGCT
ACAGGAAGCTGAGGAGCTGCTGTCTTTGGCTGTTCAAATACAGCCAGACTTTGCCGCT
GCGTGGATGAATCTAGGCATAGTGCAGAATAGCCTGAAACGGTT~rAAC~rAC~rArnr~r ATACTC
CTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCCAAA
Start: ATG at 101 ~ ~ORF Stop: TGA at 1673 SEQ ID NO. 114 ~~ 524 as y.~ MW at 59138.5kD
-_ NOV23a, MLCKEQGTTVLGLNAVFDILVIGKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTL

Protein Sequence WLCFDWSMGCIPLIKSISDWRVIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFL
VIPFLPASNLFFRVGFWAERVLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGI
LFINTLRCVLRSGEWRSEEQLFRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYRE
AVRLNPKYVHAMNNLGNILKERNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKR
FEAAEQSYRTAIKHRRKYPDCYYNLGRLYADLNRHVDALNAWRNATVLKPEHSLAWNN
MIILLDNTGNLAQAEAVGREALELIPNDHSLMFSLANVLGKSQKYKESEALFLKAIKA
~NPNAASYHGNLAVLYHRWGHLDLAKKHYEISLQLDPTASGTKENYGLLRRKLELMQKK
AV
SEQ ID NO: 115 1515 by NOV23b, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

Sequence ~CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
AAACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
TGTTTTGATTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
TAATTGCACTTGCAGCACTCTGGTTCTGCCTAATTGGCCTGATATGCCAAGCCCTGT
CTCTGAAGACGGCCACAAGAGAAGGATCCTTACTCTGGGCCTGGGATTTCTCGTTAT
CCATTTCTCCCTGCGAGTAACCTGTTCTTCCGAGTGGGCTTCGTGGTCGCGGAGCGT
TGAGCAAACATACCAAGAAAAAGAAACTCATTGCCGCTGTCGTGCTGGGAATCTTATT
CATCAACACGCTGAGATGTGTGCTGCGCAGCGGCGAGTGGCGGAGTGAGGAACAGCTT
TGGCTGATAAAGGCAACCAGACAGCTGCCATCAGATACTACCGGGAAGCTGT
AAATCCCAAGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA
TGAATCTAGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
ACTACAACCTCGGGCGTCTGTATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
GTGGAGAAATGCCACCGTGCTGAAACCAGAGCACAGCCTGGCCTGGAACAACATGAT
ATACTCCTCGACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
TGGAATTAATACCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
ATCCCAGAAATACAAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
AATGCTGCAAGTTACCATGGTAATTTGGCTGTGCTTTATCATCGTTGGGGACATCTA
ACTTGGCCAAGAAACACTATGAAATCTCCTTGCAGCTTGACCCCACGGCATCAGGAA
Start: at 1 ORF Stop: end of sequence ID NO: 116 X505 as BMW at 57228.1kD
L~OV23b, EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLP'izM'1~LL~1W c~ciAUMLrvttwrclrmmrr Protein Sequence ~VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFWAER
VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSEEQL
LQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRK
LGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAV
IPNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYHR
KKHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE
EQ ID NO: 117 1515 by OV23C, GAATTCAAATTCAATCU'1"i'L'1'UUAAH'1"1'CU'1'CLHtiljtjt~U'1't~u'1'taLta'1't~titztj~
tav~rural eauence ~CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
AACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
GTTTTGATTGGTCAATGGGCTGCACCCCCCTCATTAAGTCCATCAGCGACTGGAGG
AATTGCACTTGCAGCACTCTGGTTCTGCCTAATTGGCCTGATATGCCAAGCCCTGT
TT
TCTGTCTGTGTGTCCCCTCAATGCTAAGGTTCACTACAACATTGGCA
GATAAAGGCAACCAGACAGCTGCCATCAGATACTACCGGGAAGCTGT
CCAAGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA
ACAGGAAGCTGAGGAGCTGCTGTCTTTGGCTGTTCAAATACAGCCAG
17g ACCCAGACTGT
CGTGGAGAAATGCCACCGTGCTGAAACCAGAGCACAGCCTGGCCTGGAACAACATGAT
TATACTCCTCGACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
CTGGAATTAATACCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
AATCCCAGAAATACAAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
aanmr_rmnranr_mmnrrumrrmAATTTGGCTGTGCTTTATCATCGTTGGGGGCATCTA
AGAACTAATGCAAAAGAAAGCTGT
Start: at 1 , ~ORF Stop: end of sequence ID NO: 118 505 as ~MW at 57216.OkD
L~OV23c, EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGMLYVRWRIMGTGPP

Protein Sequence VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFVVAER
VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSEEQL
FRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYREAVRLNPKYVHAMNNLGNILKE
RNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKYPDC
YYNLGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAVGREA
LELIPNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYHRWGHL
DLAKKHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE
SEQ ID NO: 119 1515 by OV23d, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

equence ~CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
ATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
TTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
CTTGCAGCACTCTGGTTCTGCCTAATTGGCCTGATATGCCAAGCCCTGT
ACGGCCACAAGAGAAGGATCCTTACTCTGGGCCTGGGATTTCTCGTTAT
ACCTCCCCAGCGTTGGGTACTGTGTGCTGCTGACTTTTGGATTCGGAGCCC
arnmnrrnArAAAAAC~AAACTCATTGCCGCTGTCGTGCTGGGAATCTTATT
CATGAATAATCTTGGAAATATCTTAAAAGAA
TACTACAACCTCGGGCGTCTGTATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
CGTGGAGAAATGCCACCGTGCTGAAACCAGAGCACAGCCTGGCCTGGAACAACATGAT
A
ACGGTCTGCTGAGAAGAAAGCTAGAACTAATGCAAAAGAAAGCTGT
Start: at 1 ORF Stop: end of sequence SEQ ID NO: 120 505 as ~MW at 57216.OkD
OV23d, ~ EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGMLYVRWRIMGTGPP

rotein Sequence ~VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFVVAER

VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSE$QL
FRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYREAVRLNPKYVHAMNNLGNILKE
RNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKYPDC
LMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYHR
SLQLDPTASGTKENYGLLRRKLELMQKKAVLE
ID NO: 121 1515 by OV23e, ~GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

eauence CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
AAACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
TGTTTTGATTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
GCTCTGAAGACGGCCACAAGAGAAGGATCCTTACTCTGGGCCTGGGATTTCTCGTTAT
CCCATTTCTCCCTGCGAGTAACCTGTTCTTCCGAGTGGGCTTCGTGGTCGCGGAGCGT
GTCCTCTACCTCCCCAGCGTTGGGTACTGTGTGCTGCTGACTTTTGGATTCGGAGCCC
TGAGCAAACATACCAAGAAAAAGAAACTCATTGCCGCTGTCGTGCTGGGAATCTTATT
CATCAACACGCTGAGATGTGTGCTGCGCAGCGGCGAGTGGCGGAGTGAGGAACAGCTT
GATAAAGGCAACCAGACAGCTGCCATCAGATACTACCGGGAAGCTGT
CCAAGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA
ACAGGAAGCTGAGGAGCTGCTGTCTTTGGCTGTTCAAATACAGCCAG
GCGTGGATGAATCTAGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
AAAGTTACCGGACAGCAATTAAACACAGAAGGAAATACCCAGACTGT
CGGGCGTCTGTATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
GCCACCGTGCTGAAACCAGAGCACAGCCTGGCCTGGAACAACATGAT
ACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
ATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
GGTAATTTGGCTGTGCTTTATCATCGTTGGGGACATCTA
ATGAAATCTCCTTGCAGCTTGACCCCACGGCATCAGGAA
GCTGAGAAGAAAGCTAGAACTAATGCAAAAGAAAGCTGT
Start: at 1 ORF Stop: end of sequence SEQ ID NO: 122 X505 as BMW at 57222.1kD
L~OV23e, EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGMLYVRWRIMGTGPP

Protein Sequence VIALAALWFCLIGPICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFVVAER
VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSEEQL
FRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYREAVRLNPKYVHAMNNLGNILKE
RNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKYPDC
YYNLGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAVGREA
LELIPNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYHRWGHL
DLAKKHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE
EQ ID NO: 123 1515 by OV23f, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

equence ~CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
CCCCCTCATTAAGTCCATCAGCGACTGGAGG
TGCCTAATTGGCCTGATATGCCAAGCCCTGT
TCCTTACTCTGGGCCTGGGATTTCTCGTTAT
Ig~

ACCAAGAAAAAGAAACTCATTGCCGCTGTCGTGCTGGGAATCTTATT
AAGGTTCACTACAACATTGGCA
.TCAGATACTACCGGGAAGCTGT
TCTTGGAAATATCTTAAAAGAA
ATGAATCTAGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
ACCGGACAGCAATTAAACACAGAAGGAAATACCCAGACTGT
TCTGTATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
ACCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
TACAAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
GTTACCATGGTAATTTGGCTGTGCTTTATCATCGTTGGGGACATCTA
.GAAACACTATGAAATCTCCTTGCAGCTTGACCCCACGGCATCAGGAA
TACGGTCTGCTGAGAGGAAAGCTAGAACTAATGCAAAAGAAAGCTGT
CTCGAG
Start: at 1 ORF Stop: end of sequence ID N0: 124 505 as MW at 57128.9kD
L~OV2 3 f , EFKFNVLEI VQKVLHKDKSLENLC3MLIUVC~Iil~l~r~tun~l~lmwStiUtat~nL x mcmcmm i yr r Protein Sequence VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFWAER
VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSEEQL
FRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYREAVRLNPKYVHAMNNLGNILKE
RNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKYPDC
YYNLGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAVGREA
r.u-r.r~rmucr,rrtFCr,atamr,ru~nKYKRRRAT,FI~KAIKANPNAASYHGNLAVLYHRWGHL
ISLQLDPTASGTKENYGLLRGKLELMQKKAVLE
EQ ID NO: 125 1515 by L~OV23g, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

AAACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
TGTTTTGATTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
ACCTCCCCAGCGTTGGGTACTGTGTGCTGCTGACTTTTGGATTCGGAGCCC
n~nmnrrnannnnnnrAAACTCATTGCCGCTGTCGTGCTGGGAATCTTATT
CTGCGTGGATGAATCTAGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
GCAAAGTTACCGGACAGCAATTAAACACAGAAGGAAATACCCAGACTGT
CTCGGGCGTCTGTATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
ATGCCACCGTGCTGAAACCAGAGCACAGCCTGGCCTGGAACAACATGAT
CGACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
AAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
ACCATGGTAATTTGGCTGTGCTTTATCATCGTTGGGGACATCTA

CTAAGGAGAATTACGGTCTGCTGAGAAGAAAGCTAGAACTAATGCAAAAGAAAGCTGT
CCTCGAG
ORF Start: at 1 ORF Stop: end of sequence SEQ ID N0: 126 505 as ~MW at 57228.1kD
LJOV23g, EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGMLYVRWRIMUTGPP

Protein Sequence VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFVVAER
VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSEEQL
FRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYREAVRLNPKYVHAMNNLGNILKE
RNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKYPDC
YYNLGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAVGREA
LELIPNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYHRWGHL
DLAKKHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE
ID NO: 127 1515 by I~OV23h, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

Sequence CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
CAAGAAAAAGAAACTCATTGCCGCTGTCGTGCTGGGAATCTTATT
AAGGTTCACTACAACATTGGCA
AAATCCCAAGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA
AGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
ATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
ATACTCCTCGACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
TGGAATTAATACCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
n.mrrCAC~AAAmACAI~GGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
AGAAACACTATGAAATCTCCTTGCAGCTTGACCCCACGGCATCAGGAA
TTACGGTCTGCTGAGAAGAAAGCTAGAACTAATGCAAAAGAAAGCTGT
Start: at 1 ORF Stop: end of sequence SEQ ID NO: 128 X505 as BMW at 57170.1kD
t~TOV23h, EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGMLYVRWRIMGTGPP

Protein Sequence VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFWAER
VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSEEQL
QEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRR
GRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEA
PNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYH
KHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE

EQ ID N0: 129 1515 by NOV23i, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

Sequence ~CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
TAAACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
GTGTTTTGATTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
GTAATTGCACTTGCAGCACTCTGGTTCTGCCTAATTGGCCTGATATGCCAAGCCCTGT
GCTCTGAAGACGGCCACAAGAGAAGGATCCTTACTCTGGGCCTGGGATTTCTCGTTAT
CCCATTTCTCCCCGCGAGTAACCTGTTCTTCCGAGTGGGCTTCGTGGTCGCAGAGCGT
CATCAACACGCTGAGATGTGTGCTGCGCAGCGGCGAGTGGCGGAGTGAGGAACAGCTT
TTCAGAAGTGCTCTGTCTGTGTGTCCCCTCAATGCTAAGGTTCACTACAACATTGGCA
AAAACCTGGCTGATAAAGGCAACCAGACAGCTGCCATCAGATACTACCGGGAAGCTGT
AAGATTAAATCCCAAGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA
CTAGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
CAGCAATTAAACACAGAAGGAAATACCCAGACTGT
TGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
ATACTCCTCGACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
TGGAATTAATACCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
AmrrrArpAAmAe~AAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
GACTTGGCCAAGAAACACTATGAAATCTCCTCGCAGCTTGACCCCACGGCATCAGGAA
CTAAGGAGAATTACGGTCTGCTGAGAAGAAAGCTAGAACTAATGCAAAAGAAAGCTGT
Start: at 1 ORF Stop: end of sequence ID N0: 130 X505 as BMW at 57221.OkD
BTOV23i, EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGMLYVRWRIMG

Protein Sequence VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFV
~VLYLPSVGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRS
LQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKYPDC
LGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAVGREA
IPNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYRGNLAVLYHRWGHL
KKHYEISSQLDPTASGTKENYGLLRRKLELMQKKAVLE
EQ ID N0: 131 1515 by L~OV23j, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

Sequence CTCTGGAGGGGCTGGGATACTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
TAAACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
GTGTTTTGATTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
GTAATTGCACTTGCAGCACTCTGGTTCTGCCTAATTGGCCTGATATGCCAAGCCCTGT
GCTCTGAAGACGGCCACAAGAGAAGGATCCTTACTCTGGGCCTGGGATTTCTCGTTAT
CCCATTTCTCCCCGCGAGTAACCTGTTCTTCCGAGTGGGCTTCGTGGTCGCAGAGCGT
GATGTGTGCTGCGCAGCGGCGAGTGGCGGAGTGAGGAACAGCTT
GTCTGTGTGTCCCCTCAATGCTAAGGTTCACTACAACATTGGCA
AAAGGCAACCAAACAGCTGCCATCAGATACTACCGGGAAGCTGT
AGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA

ATGAGCTACAGGAAGCTGAGGAGCTGCTGTCTTTGGCTGTTCAAATACAGCCAG
TGCCGCTGCGTGGATGAATCTAGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
GCAGAGCAAAGTTACCGGACAGCAATTAAACACAGAAGGAAATACCCAGACTGT
ACAACCTCGGGCGTCTGTATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
ACTCCTCGACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
GAATTAATACCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
CCCAGAAATACAAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
TGCTGCAAGTTACCATGGTAATTTGGCTGTGCTTTATCATCGTTGGGGACATCTA
TTGGCCAAGAAACACTATGAAATCTCCTTGCAGCTTGACCCCACGGCATCAGGAA
Start: at 1 ~ORF Stop: end of sequence SEQ ID N0: 132 505 as ~MW at 57210.OkD
OV23j, EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGILYVRWRIMV

rotein Sequence ~VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFV
HYNIGKNLADKGNQTAAIRYYREAVRLNPKYVHAM
VQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAI
PNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNA
KHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE
ID N0: 133 1515 by tJOV23k, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT

Sequence CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
GCCTTCACCGAGGTGGACAACCCGGCCTCCTTTGCTGACAGCATGCTGGTGAGGGCCG
TAAACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
GTGTTTTGATTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
~rmAAmmrr.ACmmGCAGCACTCTGGTTCTGCCTAATTGGCCTGATATGCCAAGCCCTGT
CATTTCTCCCCGCGAGTAACCTGTTCTTCCGAGTGGGCTTCGTGGTCGCGGAGCGT
CCTCTACCTCCCCAGCATTGGGTACTGTGTGCTGCTGACTTTTGGATTCGGAGCCC
AGCAAACATACCAAGAAAAAGAAACTCATTGCCGCTGTCGTGCTGGGAATCTTATT
TCAACACGCTGAGATGTGTGCTGCGCAGCGGCGAGTGGCGGAGTGAGGAACAGCTT
CAGAAGTGCTCTGTCTGTGTGTCCCCTCAATGCTAAGGTTCACTACAACATTGGCA
AACCTGGCTGATAAAGGCAACCAGACAGCTGCCATCAGATACTACCGGGAAGCCGT
GATTAAATCCCAAGTATGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA
GAATGAGCTACAGGAAGCTGAGGAGCTGCTGTCTTTGGCTGTTCAAATACAGCCAG
TTTGCCGCTGCGTGGATGAATCTAGGCATAGTGCAGAATAGCCTGAAACGGTTTGA
ACTACAACCTCGGGCGTCTGTATGCAGATCTCAATCGCCACGTGGATGCCTTGAATG
CAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
CCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
ACAAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
TTACCATGGTAATTTGGCTGTGCTTTATCATCGTTGGGGACATCTA
AAACACTATGAAATCTCCTTGCAGCTTGACCCCACGGCATCAGGAA
ACGGTCTGCTGAGAAGGAAGCTAGAACTAATGCAAAAGAAAGCTGT
Start: at 1 ORF Stop: end of sequence ID N0: 134 X505 as BMW at 57242.1kD
k, ~EFKFNVLEIVQKVLHKDKSLENLGMLRNGGLLFRMTLLTSGGAGMLYVRWRIMGTGPP
1~4 rotein Sequence VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFW
VLYLPSIGYCVLLTFGFGALSKHTKKKKLIAAWLGILFINTLRCVLRSGEWRSE
FRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYREAWLNPKYVHAMNNLGNI
RNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKY
YYNLGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAVG
LELTPNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYHRW
~DLAKKHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE
EQ ID N0: 135 1515 by OV231, GAATTCAAATTCAATGTTCTGGAAATTGTCCAGAAGGTACTACATAAGGACAAGTCAT
74104491 DNA ~TAGAGAATCTCGGCATGCTCAGGAACGGGGACCTCCTCTTCAGAATGACCCTGCTCAC
arnianra CTCTGGAGGGGCTGGGATGCTCTACGTGCGCTGGAGGATCATGGGCACGGGCCCGCCG
AAACTACAATTACTACTATTCATTGAATGCCTGGCTGCTGCTGTGTCCCTGGTGGCT
TGTTTTGATTGGTCAATGGGCTGCATCCCCCTCATTAAGTCCATCAGCGACTGGAGG
manmmreneTTrc~AGCaCTCTGGTTCTGCCTAATTGGCCTGATATGCCAAGCCCTGT
AACCTGTTCTTCCGAGTGGGCTTCGTGGTCGCGGAGCGT
TTGGGTACTGTGTGCTGCTGACTTTTGGATTCGGAGCCC
TGCTAAGGTTCACTACAACATTGGCA
TGTTCATGCCATGAATAATCTTGGAAATATCTTAAAAGAA
~rTrArt~AGCTGCTGTCTTTGGCTGTTCAAATACAGCCAG
TG
TACTCCTCGACAATACAGGTAATTTAGCCCAAGCTGAAGCAGTTGGAAGAGAGGCA
GGAATTAATACCTAATGATCACTCTCTCATGTTCTCGTTGGCAAACGTGCTGGGGA
TCCCAGAAATACAAGGAATCTGAAGCTTTATTCCTCAAGGCAATTAAAGCAAATCC
ATGCTGCAAGTTACCATGGTAATTTGGCTGTGCTTTATCATCGTTGGGGACATCTA
CTTGGCCAAGAAACACTATGAAATCTCCTTGCAGCTTGACCCCACGGCATCAGGAA
AAGGAGAATTACGGTCTGCTGAGAAGAAAGCTAGAACTAATGCAAAAGAAAGCTGT
Start: at 1 iORF Stop: end of iilsequence ID N0: 136 X505 as BMW at 57300.1kD
OV231, EFKFNVLEIVQKVLHKDKSLENLGMLRNGDLLFRMTLLTSGGAGMLYVRWRIMGTGPP

rotein Sequence VIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPFLPASNLFFRVGFWAER
VLYLPSIGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINTLRCVLRSGEWRSEEQL
FRSALSVCPLNAKVHYNIGKNLADKGNQTAAIRYYREAWLNPKYVHAMNNLGNILKE
RNELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFEAAEQSYRTAIKHRRKYPDC
YYNLGRLYADLNRHVDALNAWRNATVLKPEHSLAWNNMIILLDNTGNLAQAEAVGREA
LELIPNDHSLMFSLANVLGKSQKYKESEALFLKAIKANPNAASYHGNLAVLYHRWGHL
DLAKKHYEISLQLDPTASGTKENYGLLRRKLELMQKKAVLE
ID NO: 137 855 by UTOV23iri, GAATTCAGCCiCiCaiACi'1'hliC:CiUACi'1'CiACiIiAAl:Alit:'1'~1_1_lWHlit~it~lwjwlUlwl ~yvi Sequence ~CCAGACAGCTGCCATCAGATACTACCGGGAAGCTGTAAGATTAAATCCCAAGTATGTT
CATGCCATGAATAATCTTGGAAATATCTTAAAAGAAAGGAATGAGCTACAGGAAGCTG

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
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Claims (45)

What is claimed is:

1. An isolated polypeptide comprising the mature form of an amino acid sequenced selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 141.

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 141.

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 141.

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 141.

5. The polypeptide of claim 1 wherein said polypeptide is naturally occurring.

6. A composition comprising the polypeptide of claim 1 and a carrier.

7. A kit comprising, in one or more containers, the composition of claim 6.

8. The use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease selected from a pathology associated with the polypeptide of claim 1, wherein the therapeutic comprises the polypeptide of claim 1.

9. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:

(a) providing said sample;
(b) introducing said sample to an antibody that binds immunospecifically to the polypeptide; and (c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.

10. A method for determining the presence of or predisposition to a disease associated with altered levels of expression of the polypeptide of claim 1 in a first mammalian subject, the method comprising:
a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and b) comparing the expression of said polypeptide in the sample of step (a) to the expression of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease, 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 N0:2n, wherein n is an integer between 1 and 141 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 N0:2n-1, wherein n is an integer between 1 and 141.

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

22. A nucleic acid molecule, wherein the nucleic acid molecule 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 141.

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 JD
NO:2n, wherein n is an integer between 1 and 141.

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 141.

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 141, or a complement of said nucleotide sequence.

26. A vector comprising the nucleic acid molecule of claim 20.

27. The vector of claim 26, further comprising a promoter operably linked to said nucleic acid molecule.

28. A cell comprising the vector of claim 26.

29. An antibody that immunospecifically binds to the polypeptide of claim 1.

30. The antibody of claim 29, wherein the antibody is a monoclonal antibody.

31. The antibody of claim 29, wherein the antibody is a humanized antibody.

32. A method for determining the presence or amount of the nucleic acid molecule of claim 20 in a sample, the method comprising:
(a) providing said sample;
(b) introducing said sample to a probe that binds to said nucleic acid molecule;
and (c) determining the presence or amount of said probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.

33. The method of claim 32 wherein presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type.

34. The method of claim 33 wherein the cell or tissue type is cancerous.

35. A method for determining the presence of or predisposition to a disease associated with altered levels of expression of the nucleic acid molecule of claim 20 in a first mammalian subject, the method comprising:
a) measuring the level of expression of the nucleic acid in a sample from the first mammalian subject; and b) comparing the level of expression of said nucleic acid in the sample of step (a) to the level of expression of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease;
wherein an alteration in the level of expression of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

36. A method of producing the polypeptide of claim 1, the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and 141.

37. The method of claim 36 wherein the cell is a bacterial cell.

38. The method of claim 36 wherein the cell is an insect cell.

39. The method of claim 36 wherein the cell is a yeast cell.

40. The method of claim 36 wherein the cell is a mammalian cell.

41. A method of producing the polypeptide of claim 2, the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and 141.

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.