Definitions

• the present itivention relates to novel antibodies that bind immunospecifically to antigenic polypeptides, wherein the polypeptides have characteristic properties related to biochemical or physiological responses in a cell, a tissue, an organ or an organism.
• the novel polypeptides are gene products of novel genes, or are specified biologically active fragments or derivatives thereof.
• Methods of use of the antibodies encompass procedures for diagnostic and prognostic assay of the polypeptides, as well as methods of treating diverse pathological conditions.
• Eukaryotic cells are characterized by biochemical and physiological processes which under normal conditions are extremely highly balanced to achieve the preservation and propagation of the cells.
• the regulation of the biochemical and physiological processes involves intricate signaling pathways.
• signaling pathways involve extracellular signaling proteins, cellular receptors that bind the signaling proteins, and signal transducing components located within the cells.
• Signaling proteins may be classified as endocrine effectors, paracrine effectors or autocrine effectors.
• Endocrine effectors are signaling molecules secreted by a given organ into the circulatory system, which are then transported to a distant target organ or tissue.
• the target cells include the receptors for the endocrine effector, and when the endocrine effector binds, a signaling cascade is induced.
• Paracrine effectors involve secreting cells and receptor cells in close proximity to each other, for example two different classes of cells in the same tissue or organ. One class of cells secretes the paracrine effector, which then reaches the second class of cells, for example by diffusion through the extracellular fluid.
• the second class of cells contains the receptors for the paracrine effector; binding of the effector results in induction of the signaling cascade that elicits the corresponding biochemical or physiological effect.
• Autocrine effectors are highly analogous to paracrine effectors, except that the same cell type that secretes the autocrine effector also contains the receptor. Thus the autocrine effector binds to receptors on the same cell, or on identical neighboring cells. The binding process then elicits the characteristic biochemical or physiological effect.
• Signaling processes may elicit a variety of effects on cells and tissues including by way of nonlimiting example induction of cell or tissue proliferation, suppression of growth or proliferation, induction of differentiation or maturation of a cell or tissue, and suppression of differentiation or maturation of a cell or tissue.
• 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.
• the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
• novel nucleic acids and polypeptides are referred to herein as NO X, or NOV1, NOV2, NOV3, etc., nucleic acids and polypeptides.
• NOVX nucleic acid or polypeptide sequences.
• the invention provides an isolated polypeptide comprising a mature form of a NOVX amino acid.
• the polypeptide can be, for example, a NOVX amino acid sequence or a variant of a NOVX amino acid sequence, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so change ⁇ .
• the invention also includes fragments of any of NOVX polypeptides.
• the invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. Also included in the invention is a NOVX polypeptide that is a naturally occurring variant of a NOVX sequence.
• the variant includes an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a NOVX nucleic acid sequence.
• the NOVX polypeptide is a variant polypeptide described therein, wherein any amino acid specified in the chosen sequence is changed to provide a conservative substitution.
• invention provides a method for determining the presence or amount of the NOVX polypeptide in a sample by providing a sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the NOVX polypeptide, thereby determining the presence or amount of the NOVX polypeptide in the sample.
• the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide in a mammalian subject by measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and comparing the amount of the polypeptide in the sample of the first step to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, the disease.
• An alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
• the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier.
• the therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide.
• the invention includes, in one or more containers, a therapeutically- or prophylactically- effective amount of this pharmaceutical composition.
• the invention provides the use of a therapeutic in the manxrfacture of a medicament for treating a syndrome associated with a human disease that is associated with a NOVX polypeptide.
• the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample expressing the NOVX polypeptide with antibody that binds the NOVX polypeptide in an amount sumc ⁇ ent to modulate toe activity of the polypeptide.
• the invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof.
• the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.
• the nucleic acid encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.
• the nucleic acid molecule differs by a single nucleotide from a NOVX nucleic acid sequence.
• the NOVX nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 73, or a complement of the nucleotide sequence.
• the invention provides a nucleic acid molecule wherein the nucleic acid includes the nucleotide sequence of a naturally occurring allelic nucleic acid variant. Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein. The invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
• the invention provides for a method for determining the presence or amount of a nucleic acid molecule in a sample by contacting a sample with a probe that binds a NOVX nucleic acid and determining the amount of the probe that is bound to the NOVX nucleic acid.
• the NOVX nucleic may be a marker for cell or tissue type such as a cell or tissue type that is cancerous.
• the invention provides a method for determining the presence of or predisposition to a disease associated with altered levels of a nucleic acid molecule in a first mammalian subject, wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
• the invention further provides an antibody that binds immunospecifically to a NOVX polypeptide.
• the NOVX antibody may be monoclonal, humanized, or a fully human antibody.
• the antibody has a dissociation constant for the binding of the NOVX polypeptide to the antibody less than 1 x 10 "9 M.
• the NOVX antibody neutralizes the activity of the NOVX polypeptide.
• the invention provides for tlie use ot a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, associated with a NOVX polypeptide.
• the therapeutic is a NOVX antibody.
• 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.
• the present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences, their encoded polypeptides, antibodies, and other related compunds. The sequences are collectively referred to herein as "NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any of the novel sequences disclosed herein. Table 1 provides a summary of the NOVX nucleic acids and their encoded polypeptides. TABLE 1. NOVX Polynucleotide and Polypeptide Sequences and Corresponding SEQ ID Numbers
• Table 1 indicates the homology of NOVX polypeptides to known protein families.
• nucleic acids and polypeptides, antibodies and related compounds according to the invention corresponding to a NOVX as identified in column 1 of Table 1 will be useful in therapeutic and diagnostic applications implicated in, for example, pathologies and disorders associated with the known protein families identified in column 5 of Table 1.
• NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
• the various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
• 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.
• the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit diseases associated with the protein families listed in Table 1.
• 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.
• 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.
• 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 research tools. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) a biological defense weapon.
• 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 73; (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 73, 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 73; (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 73 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 residue
• 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 73; (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 73 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 73; (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 73, in which any amino acid specified in the chosen sequence is changed
• 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-l, wherein n is an integer between 1 and 73; (b) a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 73 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-l, wherein n is an integer between 1 and 73; and (d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected
• One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX- encoding nucleic acids (e.g., NOVX mRNA's) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules.
• nucleic acid fragments sufficient for use as hybridization probes to identify NOVX- encoding nucleic acids (e.g., NOVX mRNA's) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules.
• 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
• DNA molecules e.g., cDNA or genomic DNA
• RNA molecules e.g., mRNA
• analogs of the DNA or RNA generated using nucleotide analogs e.g., mRNA
• 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.
• a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
• the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
• the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
• Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.
• 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.
• a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining.
• a "mature" form of a polypeptide or protein may arise from a step of post- translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristylation or phosphorylation.
• a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
• probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
• isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
• an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
• 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.).
• an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
• a nucleic acid molecule of the invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:2 «-l, wherein n is an integer between 1-73, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
• NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et ⁇ l., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ l., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
• a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
• the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
• oligonucleotides corresponding to NOVX ' hucieotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
• oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
• a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
• Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
• an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NO:2r ⁇ -l, wherein n is an integer between 1-73, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
• an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence SEQ ID NO:2 «-l, wherein n is an integer between 1-73, 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 NO:2 «-l, wherein n is an integer between 1-73, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO:2 «-l, wherein n is an integer between 1-73, that it can hydrogen bond with little or no mismatches to the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1-73, thereby forming a stable duplex.
• 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.
• Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, " and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
• a full-length NOVX clone is identified as containing an ATG translation start codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence lacking an ATG start codon therefore encodes a truncated C-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 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.
• Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
• Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 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 aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and
• a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
• Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Tsofofms 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.
• homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
• homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
• a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein.
• Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:2/z-l, wherein n is an integer between 1-73, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.
• a NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX nucleic acid.
• An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide.
• a stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.
• An ORF that represents the coding sequence for a full protein begins with an ATG "start” codon and terminates with one of the three “stop” codons, namely, TAA, TAG, or TGA.
• an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both.
• 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 NO:2 «- 1, wherein n is an integer between 1-73; or an anti-sense strand nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1-73; or of a naturally occurring mutant of SEQ ID NO:2 «-l, wherein n is an integer between 1-73.
• Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
• the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
• the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
• Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
• a polypeptide having a biologically-active portion of a NOVX polypeptide refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
• a nucleic acid fragment encoding a "biologically- active portion of NOVX” can be prepared by isolating a portion of SEQ ID NO:2n-l, wherein n is an integer between 1 -73, 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.
• the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID NO:2 «-l, wherein n is an integer between 1-73, 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:2 «-l, wherein n is an integer between 1-73.
• an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO:2 «, wherein n is an integer between 1-73.
• NOVX nucleotide sequences of SEQ ID NO:2 «-l, wherein n is an integer between 1-73
• 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.
• the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein.
• Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from any one of the human SEQ ID NO:2 «-l, wherein n is an integer between 1-73, 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.
• an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2r ⁇ -l , wherein n is an integer between 1-73.
• the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.
• an isolated nucleic acid molecule of the invention hybridizes to the coding region.
• the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
• Homologs i.e., nucleic acids encoding NOVX proteins derived from species other than human
• other related sequences e.g., paralogs
• stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
• Tm thermal melting point
• ' 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.
• 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.
• 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.
• 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).
• a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1-73, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided.
• 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 55 °C, followed by one or more washes in IX 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.
• low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl ' (pH T.5J, 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg ml denatured salmon sperm DNA, 10% (wt/volt) dextran sulfate at 40 °C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50 °C.
• Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
• nucleotide sequences of SEQ ID NO:2 ⁇ ?-l wherein n is an integer between 1 -73, thereby leading to changes in the amino acid sequences of the encoded NOVX proteins, without altering the functional ability of said NOVX proteins.
• nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:2/ , wherein n is an integer between 1-73.
• 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.
• amino acid residues that are conserved among the NOVX proteins of the invention are particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
• Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from any one of SEQ ID NO:2 ⁇ -l, wherein n is an integer between 1-73, yet retain biological activity.
• the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID NO:2 «, wherein n is an integer between 1-73.
• the protein encoded by the nucleic acid molecule is at least about 60%> homologous to SEQ ID NO:2«, wherein n is an integer between 1-73; more preferably at least about 70% homologous to SEQ ID NO:2«, wherein n is an integer between 1-73; still more preferably at least about 80% homologous to SEQ ID NO:2/ ⁇ , whe ein w is an integer Between 1-73; even more preferably at least about 90% homologous to SEQ ID NO:2 «, wherein n is an integer between 1-73; and most preferably at least about 95% homologous to SEQ ID NO:2r ⁇ , wherein n is an integer between 1-73.
• An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID NO:2 «, wherein n is an integer between 1-73 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:2r ⁇ -l, wherein n is an integer between 1-73, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into any of SEQ ID NO:27 -l, wherein n is an integer between 1-73, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• 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.
• 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
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family.
• mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity.
• 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.
• 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.
• a mutant NOVX protein can be assayed for (i) the ability to form protein ⁇ rotein 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 (Hi) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
• a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
• Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2 «-l , wherein n is an integer between 1 -73, 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).
• antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof.
• an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a NOVX protein.
• coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
• the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein.
• noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
• antisense nucleic acids of the invention can'be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
• the antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA.
• 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.
• an antisense nucleic acid e.g., an antisense oligonucleotide
• an antisense nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
• modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methyl guanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomefhyl-2-thiouracil, beta-D-mannosylqueosine,
• the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
• the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation).
• the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
• An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
• antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
• 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.
• 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.
• the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
• An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -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., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., Inoue, et al, 1987. FEBSLett. 215: 327-330.
• 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.
• 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.
• ribozymes e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591
• a ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX cDNA disclosed herein (i. e. , any one of SEQ ID NO:2 ⁇ ?-l , wherein n is an integer between 1-73).
• 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.
• 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.
• nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid e.g., the NOVX promoter and/or enhancers
• 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.
• the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, 1996. Bioorg Med Chem 4: 5-23.
• peptide nucleic acids 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.
• PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
• PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directedTPCR clamping; as artiiiciai restriction enzymes when used in combination with other enzymes, e.g., Sj 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.
• PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
• PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
• PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleotide bases, and orientation (see, Hyrup, et al., 1996. supra).
• the synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363.
• a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al, 1996. supra.
• chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1915. Bioorg. Med. Chem. Lett. 5: 1119-11124.
• 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. WO88/09810) or the blood-brain barrier (see, e.g. , PCT Publication No. WO 89/10134).
• peptides e.g., for targeting host cell receptors in vivo
• 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
• oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g.,
• 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.
• 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 NO:2«, wherein n is an integer between 1-73.
• the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in any one of SEQ ID NO:2 «, wherein n is an integer between 1-73, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
• 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.
• native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
• NOVX proteins are produced by recombinant DNA techniques.
• 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.
• 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.
• non-NOVX proteins also referred to herein as a "contaminating protein”
• 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.
• 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 NO:2n, wherein n is an integer between 1-73) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of a NOVX protein.
• 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.
• 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.
• the NOVX protein has an amino acid sequence of SEQ ID NO:2r ⁇ , wherein n is an integer between 1-73.
• the NOVX protein is substantially homologous to SEQ ID NO:2rc, wherein n is an integer between 1-73, and retains the functional activity of the protein of SEQ ID NO:2rc, wherein n is an integer between 1-73, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
• the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2rc, wherein n is an integer between 1-73, and retains the functional activity of the NOVX proteins of SEQ ' ⁇ E) NO:2 «, wherein n is an integer between 1-73.
• 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.
• 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. J Mol Biol 48: 443-453.
• GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48: 443-453.
• GAP creation penalty of 5.0 and GAP extension penalty of 0.3 the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence of SEQ ID NO:2 «-l, wherein n is an integer between 1-73.
• 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.
• 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.
• substantially identical 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 a ⁇ least 99 percent sequence identity as compared to a reference sequence over a comparison region.
• the invention also provides NOVX chimeric or fusion proteins.
• NOVX chimeric or fusion proteins.
• NOVX "chimeric protein” or “fusion protein” comprises a NOVX polypeptide operatively- linked to a non-NOVX polypeptide.
• An "NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a NOVX protein of SEQ ID NO:2 ⁇ , wherein n is an integer between 1-73, 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.
• a NOVX fusion protein comprises at least one biologically- active portion of a NOVX protein.
• a NOVX fusion protein comprises at least two biologically-active portions of a NOVX protein.
• a NOVX fusion protein comprises at least three biologically-active portions of a NOVX 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.
• 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.
• GST glutthione S-transferase
• Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
• the fusion protein is a NOVX protein containing a heterologous signal sequence at its N-terminus.
• expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
• the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family.
• the NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a NOVX ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo.
• the NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival.
• 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.
• the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and different
• 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.
• DNA fragments coding for the different pol peptide 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.
• the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
• PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
• anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
• 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.
• the invention also pertains to variants of the NOVX proteins that function as either NOVX agonists ( . 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.
• 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.
• 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.
• 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.
• 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 Sj nuclease, and ligating the resulting fragment library into an expression vector.
• expression libraries can be derived which encodes N-termmal and internal fragments of various sizes of the NOVX proteins.
• Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
• antibody refers to immunoglobulin molecules and immunologically active portions of immxmoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
• Ig immxmoglobulin
• Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F ab , F ab ' and F (ab')2 fragments, and an F ab expression library.
• 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.
• 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"res ⁇ dues of the ammo acid sequence of the full length protein, such as an amino acid sequence of SEQ ID NO:2 «, wherein n is an integer between 1-73, 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.
• 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.
• at least one epitope encompassed by the antigenic peptide is a region of NOVX that is located on the surface of the protein, e.g., a hydrophilic region.
• a hydrophobicity analysis of the human NOVX protein sequence will indicate which regions of a NOVX polypeptide are particularly hydrophilic and, therefore, encode surface residues useful for targeting antibody production.
• 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.
• epitope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
• Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
• a NOVX polyppeptide or a fragment thereof comprises at least one antigenic epitope.
• An anti-NOVX antibody of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (KD) is ⁇ 1 ⁇ M, 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.
• KD equilibrium binding constant
• a protein of the invention, or a derivative, fragment, analog, nomolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
• 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.
• the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
• 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.
• 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., lysolecifhin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
• Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
• the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is disct ⁇ ssecl," ⁇ or example, ' by ' D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
• MAb monoclonal antibody
• CDRs complementarity determining regions
• Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
• 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.
• the lymphocytes can be immunized in vitro.
• the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
• 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.
• 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.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
• 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 rase " 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.
• the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
• RIA radioimmunoassay
• ELISA enzyme-linked immunoabsorbent assay
• 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.
• 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.
• the DNA can be placed into expression vectors, which are then transfected into host celts such as simian CO ' S ' 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.
• host celts such as simian CO ' S ' cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein
• 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 (U.S. Patent No.
• 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.
• 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 immxmoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
• Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et at. ' , 1988; and Pres ⁇ a, Curr. Op. Struct. Biol., 2:593-596 (1992)).
• Fc immunoglobulin constant region
• Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
• Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see 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).
• 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)).
• 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.
• 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.
• 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.
• 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.
• nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
• This animal produces B cells which secrete fully human immunoglobulins.
• the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
• 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.
• U.S. Patent No. 5,939,598 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.
• 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).
• methods can be adapted for the construction of F a expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
• Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F (ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F (a ')2 fragment; (iii) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
• Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
• one of the binding specificities is for an antigenic protein of the invention.
• the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
• 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 often 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 can be fused to immunoglobulin constant domain sequences.
• the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
• DNAs encoding the immunoglobulin heavy-c ⁇ am fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
• 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.
• 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).
• Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 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.
• 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
• 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 fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and V domains of one fragment are forced to pair with the complementary V L and VH domains of another fragment, thereby forming two antigen-binding sites.
• VH heavy-chain variable domain
• V L light-chain variable domain
• 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.
• trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
• bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.
• an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RLII (CD 16) 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.
• 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.
• 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 are also within the scope of the present invention.
• Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV nfectidh (WO ' 9T/00360; WO ' 92/200373; EP 03089).
• the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
• 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.
• cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
• the homodimeric antibody thus generated can have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
• Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560- 2565 (1993).
• 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).
• the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
• a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
• Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricofhecenes.
• a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 13 I I, I31 In, 90 Y, and ' 6 Re.
• Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3 -(2 -pyridyl dithiol) 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 l,5-difluoro-2,4-dinitrobenzene).
• SPDP N-succinimi
• aricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
• Carbon-14- labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
• the antibody in another embodiment, 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.
• a "receptor” such streptavidin
• a "ligand” e.g., avidin
• the antibodies disclosed herein can also be formulated as immunoliposomes.
• Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
• Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
• Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al .,_J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
• a chemotherapeutic agent such as Doxorubicin
• Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
• antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain are utilized as pharmacologically-active compounds (see below).
• An antibody specific for a protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein (e.g. , in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein. Antibodies directed against the protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i. e.
• detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example of a luminescent material includes luminol;
• bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1 5 1, 131 1, 35 S or 3 H.
• Antibodies of the invention may be 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.
• administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds.
• the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule.
• the receptor mediates a signal transduction pathway for which ligand is responsible.
• 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.
• 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.
• 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 " : TEe 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.
• the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred.
• 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.
• 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.
• the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
• cytotoxic agent 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.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
• 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.
• copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate non- degradable ethyl ene-vinyl acetate, degradable lactic the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
• polymers such as ethyl ene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
• 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
• labeling 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.
• 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.
• 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 in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.
• In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
• In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Thory of Enzyme Immunoassays", P.
• in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody.
• the antibody can be labeled with a radioactive marKer whose presence and location in a subject can be detected by standard imaging techniques.
• vectors preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
• viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
• vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• Other vectors e.g. , non-episomal mammalian vectors
• vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors".
• expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
• plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
• 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.
• "operably- linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
• regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
• the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
• the recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells.
• NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
• the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
• 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 (Hi) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
• 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.
• 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.
• GS 1 glutathione S-transferase
• E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET l id (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.
• the NOVX expression vector is a yeast expression vector.
• yeast Saccharomyces cerivisae examples include pYepSecl (Baldari, et al., 1987. EMBOJ. 6: 229-234), pMFa (Kurjan and Herskowifz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif).
• NOVX can be expressed in insect cells using baculovirus expression vectors.
• Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-21 5) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
• a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
• mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBOJ. 6: 187-195).
• the expression vector's control functions are often provided by viral regulatory elements.
• commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
• 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.
• suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987.
• lymphoid-specific promoters Calame and Eaton, 1988. Adv. Immunol. 43: 235-275
• promoters of T cell receptors Winoto and Baltimore, 1989.
• EMBO J. 8: 729-733 promoters of T cell receptors
• immunoglobulins Bonerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748
• neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.
• pancreas-specific promoters Eslund, et al, 1985. Science 230: 912-916
• 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 Grass, 1990. Science 249: 374-379) and the ⁇ -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.
• a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
• Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced.
• host cell and “recombinant host cell” are used interchangeably hereim If ⁇ s 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.
• 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.
• 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, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or eleetroporation.
• 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.
• 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.
• selectable markers include those that confer resistance to drugs, such as G418, 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.
• 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.
• the invention further provides methods for producing NOVX protein using the host cells of the invention.
• 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.
• the method further comprises isolating NOVX protein from the medium or the host cell.
• the host cells of the invention can also be used to produce non-human transgenic animals.
• 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 and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity.
• a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
• Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
• a transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
• 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, retro viral 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 NO:2n-l, wherein n is an integer between 1-73, can be introduced as a transgene into the genome of a non-human animal.
• a non-human homologue of the human NOVX gene such as a mouse NOVX gene
• a non-human homologue of the human 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 sequence(s) can be operably-linked to the NOVX transgene to direct express ⁇ offbf NOVX jjr ⁇ te ⁇ t ⁇ ' part ⁇ cu ⁇ ar " cells.
• transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene- encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
• a vector 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:2 -l, wherein n is an integer between 1-73), but more preferably, is a non-human homologue of a human NOVX gene.
• a mouse homologue of human NOVX gene of SEQ ID NO:2 ⁇ -l can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome.
• 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).
• 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).
• 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.
• flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
• flanking DNA both at the 5'- and 3'-termini
• the vector is ten introduced intd 1 ari"'emb ' ry ⁇ h ⁇ c " stefrrceudihe (e.g. , by eleetroporation) and cells in which the introduced NOVX gene has homologously- recombined with the endogenous NOVX gene are selected.
• the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
• an animal e.g., a mouse
• aggregation chimeras See, e.g., Bradley, 1987.
• 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. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
• transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
• a system is the cre/loxP recombinase system of bacteriophage PI .
• cre/loxP recombinase system See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236.
• Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355.
• mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
• Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
• Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813.
• a cell e.g., a somatic cell
• 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 one of the animal from which the cell (e.g., the somatic cell) is isolated.
• compositions suitable for administration 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.
• pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
• Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
• the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
• a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
• Solutions or suspensions used for parenteral, 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 bisulfite; 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.
• 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.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.
• 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.
• 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
• 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.
• 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.
• 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
• 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.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• 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.
• 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.
• suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
• retention enemas for rectal delivery.
• 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.
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
• Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
• the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
• Gene therapy vectors can be delivered to a 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.
• the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
• compositions can be included in a container, pack, or dispenser together with instructions for administration.
• 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.
• 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.
• the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity.
• 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.
• 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.
• 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.
• 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 NOV
• the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a
• 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.
• biological libraries are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.
• a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
• Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
• Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
• 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.
• test compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
• 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.
• the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.
• 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.
• 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.
• a NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g.
• 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.
• a reporter gene comprising a NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
• a cellular response for example, cell survival, cellular differentiation, or cell proliferation.
• 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.
• 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 ot 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.
• 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.
• 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/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.
• 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.
• 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-l 14, Thesit ® , Isotridecypoly(ethylene glycol ether) n , N-dodecyl ⁇ N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l- ⁇ ropane sulfonate (CHAPSO).
• non-ionic detergents such as n-oct
• binding of a tesf compouhdt ⁇ 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.
• a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
• GST- NO VX 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.
• 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
• Methods for detecting such 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.
• 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 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.
• 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. Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, et al, 1993.
• NOVX-binding proteins proteins that bind to or interact with NOVX
• NOVX-binding proteins proteins that bind to or interact with NOVX
• 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.
• a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait” and the “prey” proteins are able to interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
• a reporter gene e.g., LacZ
• the invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein. Detection Assays
• cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents.
• these sequences can be used to: (z) 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 (Hi) aid in forensic identification of a biological sample.
• this sequence can be used to map the location of the gene on a chromosome.
• This process is called chromosome mapping.
• portions or fragments of the NOVX sequences of SEQ ID NO:2 «-l, wherein n is an integer between 1-73, 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.
• NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals
• human and mouse cells As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al, 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
• PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.
• Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
• Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle.
• the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
• the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
• clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
• 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
• 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.
• the physical position of the sequence on the chromosome can be correlated with genetic map data.
• genetic map data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library).
• the relationship between genes and disease, mapped to the same chromosomal region can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al, 1987. Nature, 325: 783-787.
• differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene can be determined.
• a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
• the NOVX sequences of the invention can also be used to identify individuals from minute biological samples.
• 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).
• 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.
• 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).
• SNPs single nucleotide polymorphisms
• RFLPs restriction fragment length polymorphisms
• 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 NO:2n-l, wherein n is an integer between 1-73, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
• the invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmaco genomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
• 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.
• a biological sample e.g., blood, serum, cells, tissue
• the disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
• the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in a NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
• Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
• Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)
• 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.
• 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.
• a compound or an agent capable of detecting NOVX protein or nucleic acid e.g., mRNA, genomic DNA
• An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA.
• the nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NO:2 «-l, wherein n is an integer between 1-73, 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.
• n is an integer between 1-73
• a portion thereof such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA.
• Other suitable probes for use in the diagnostic assays of the invention are described herein.
• An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label.
• Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or
• F(ab') 2 F(ab') 2
• 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.
• 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.
• 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.
• the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
• 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.
• in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody.
• the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
• the biological sample contains protein molecules from the test subject.
• 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.
• 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.
• 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 kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard.
• the compound or agent can be packaged in a suitable container.
• the kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
• 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.
• 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.
• the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
• 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.
• a test sample refers to a biological sample obtained from a subject of interest.
• a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
• 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.
• an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
• agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
• the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).
• 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.
• 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.
• 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; (Hi) 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.
• a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
• any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
• detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al, 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994. Proc. Natl 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.
• PCR polymerase chain reaction
• LCR ligation chain reaction
• 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.
• nucleic acid e.g., genomic, mRNA or both
• 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 ⁇ 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.
• mutations in a NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
• 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.
• 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.
• genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al, 1996. Human Mutation 1: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759.
• genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al, supra.
• 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.
• any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the corresponding wild-type (control) sequence.
• Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al, 1995.
• Biotechniques 19: 448 including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl. Biochem. Biotechnol. 38: 147-159).
• RNA/RNA or RNA/DNA heteroduplexes 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.
• 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.
• RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Si nuclease to enzymatically digesting the mismatched regions.
• 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.
• control DNA or RNA can be labeled for detection.
• 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.
• DNA mismatch repair enzymes
• the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662.
• a probe based on a NOVX sequence e.g., a wild-type NOVX sequence
• 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.
• alterations in electrophoretic mobility will be used to identify mutations in NOVX genes.
• SSCP single strand conformation polymorphism
• 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.
• the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 1: 5.
• the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGG ⁇ ).
• DGG ⁇ denaturing gradient gel electrophoresis
• DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
• a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem.
• oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Natl. Acad. Sci USA 86: 6230.
• Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
• Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238).
• amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
• the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a NOVX gene.
• any cell type or tissue preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein.
• any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
• disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
• the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
• 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
• 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 agent(s) for therapeutic or prophylactic treatment of the individual.
• Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drag disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266.
• 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.
• G6PD glucose-6-phosphate dehydrogenase
• the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
• drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome pregnancy zone protein precursor enzymes CYP2D6 and CYP2C19
• NAT 2 N-acetyltransferase 2
• CYP2D6 and CYP2C19 cytochrome pregnancy zone protein precursor enzymes
• CYP2D6 and CYP2C19 cytochrome pregnancy zone protein precursor enzymes
• These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
• 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 drag response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
• 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 agent(s) for therapeutic or prophylactic treatment of the individual.
• pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
• Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX can be applied not only in basic drug screening, but also in clinical trials.
• agents e.g., drugs, compounds
• 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.
• 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.
• the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
• 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.
• an agent e.g., compound, drug or small molecule
• NOVX activity e.g., identified in a screening assay as described herein
• 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 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.
• 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.
• the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of ( ) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a NOVX protein, mRNA, or genomic DNA in the preadministration sample; (Hi) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
• an agent e.g.,
• 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.
• 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.
• the invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NOVX expression or activity.
• the disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn
• 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; (Hi) nucleic acids encoding an aforementioned peptide; (fv) 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.
• modulators i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention
• modulators i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention
• Therapeutics that increase (i.e., are agonists to) activity may be administered in a therapeutic or prophylactic manner.
• Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
• Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
• tissue sample e.g., from biopsy tissue
• assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
• Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
• immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
• hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
• 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.
• 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.
• the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell.
• An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX peptidomimetic, or other small molecule.
• 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.
• the agent inhibits one or more NOVX protein activity.
• 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, in vivo (e.g., by administering the agent to a subject).
• 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.
• 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.
• an agent e.g., an agent identified by a screening assay described herein
• 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 sftwations in which NOVX is abnormally downregulated and/or in which increased NOVX activity has a beneficial effect.
• a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders).
• a gestational disease e.g., preclampsia
• 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 vitro assays may be performed with representative cells of the type(s) 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.
• suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
• any of the animal model system known in the art may be used prior to administration to human subjects.
• NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
• 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.
• the compositions of the invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immxxne disorders, hematopoietic disorders, and the various dyslipidemias.
• Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
• a further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess antibacterial properties).
• These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
• Example A Polynucleotide and Polypeptide Sequences, and Homology Data
• the NOVl clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 1 A.
• PSort 0.3600 probability located in mitochondrial matrix space; 0.3000 probability analysis: located in microbody (peroxisome); 0.1000 probability located in lysosome (lumen); 0.0000 probability located in endoplasmic reticulum (membrane)
• NOVla protein was found to have homology to the proteins shown in the BLASTP data in Table ID.
• the NOV2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 2A.
• PSort 0.6000 probability located in nucleus; 0.3000 probability located in microbody analysis: (peroxisome); 0.2000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in mitochondrial inner membrane
• NOV2a protein was found to have homology to the proteins shown in the BLASTP data in Table 2D.
• the NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.
• PSort 0.8200 probability located in endoplasmic reticulum (membrane); 0.1900 analysis: probability located in plasma membrane; 0.1000 probability located in endoplasmic reticulum (lumen); 0.1000 probability located in outside
• NOV3a protein was found to have homology to the proteins shown in the BLASTP data in Table 3D.
• the NO 4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.
• NOV4a protein was found to have homology to the proteins shown in the BLASTP data in Table 4D.
• NOV5 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 5A. Table 5A. NOV5 Sequence Analysis
• NOV5a protein was found to have homology to the proteins shown in the BLASTP data in Table 5D.
• the NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.
• NOV6a protein was found to have homology to the proteins shown in the BLASTP data in Table 6E.
• the NOV7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 7A.
• NOV7a protein was found to have homology to the proteins shown in the BLASTP data in Table 7D.
• the NOV8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.
• PSort 0.8700 probability located in nucleus; 0.8500 probability located in analysis: endoplasmic reticulum (membrane); 0.7900 probability located in plasma membrane; 0.3325 probability located in microbody (peroxisome)
• AAY82530 Human neurotransmitter associated 1034..1211 82/194 (42%) le-32 protein sequence SEQ ID NO: 6 - 16..207 105/194 (53%) Homo sapiens, 210 aa. [WO200012685-A2, 09-MAR- 2000]
• AAY54373 cDNA sequence encoding the 36-568 120/537 (22%) 6e-23 human minor vault protein pi 93 - 644..1090 214/537 (39%) Homo sapiens, 1724 aa. [WO9962547-A1, 09-DEC-1999]
• NOV ⁇ a protein was found to have homology to the proteins shown in the BLASTP data in Table 8D.
• the NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.
• NOV9a Residues/ Identities/
• NOV9b 9..275 241/268 (89%) 1..268 241/268 (89%)
• PSort j 0.7900 probability located in plasma membrane; 0.3000 probability located in analysis: Golgi body; 0.2000 probability located in endoplasmic reticulum (membrane); j 0.1000 probability located in mitochondrial inner membrane
• NOV9a protein was found to have homology to the proteins shown in the BLASTP data in Table 9E.
• the NOV10 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 10A.
• NOVlOa protein was found to have homology to the proteins shown in the BLASTP data in Table 10D.
• the NOVl 1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11 A.
• PSort 0.8500 probability located in endoplasmic reticulum (membrane); 0.6000 analysis: probability located in nucleus; 0.4400 probability located in plasma membrane; 0.2323 probability located in microbody (peroxisome)
• NOVl 1 a protein was found to have homology to the proteins shown in the BLASTP data in Table 1 ID.
• the NOVl 2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 12 A.
• PSort 0.7000 probability located in plasma membrane; 0.3000 probability located in analysis: microbody (peroxisome); 0.2000 probability located in endoplasmic reticulum (membrane); 0.1000 probability located in mitochondrial inner membrane
• NOVl 2a protein was found to have homology to the proteins shown in the BLASTP data in Table 12E.
• the NOVl 3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 13 A.
• NOV13a Residues/ Identities/
• NOVl 3b 1..86 86/86 (100%) 31..116 86/86 (100%)
• NOVl 3a protein was found to have homology to the proteins shown in the BLASTP data in Table 13E.
• the NOVl 4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 14A.
• NOVl 4a protein was found to have homology to the proteins shown in the BLASTP data in Table 14D.
• the NOVl 5 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 15 A.
• NOV15a Residues/ Identities/
• NOVl 5a protein was found to have homology to the proteins shown in the BLASTP data in Table 15E.
• the NOVl 6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 16A.
• NOVl 6a protein was found to have homology to the proteins shown in the BLASTP data in Table 16D.
• the NOVl 7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 17A.
• PSort 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400 analysis: probability located in plasma membrane; 0.4600 probability located in Golgi body; 0.1000 probability located in endoplasmic reticulum (lumen)
• NOVl 7a protein was found to have homology to the proteins shown in the BLASTP data in Table 17E.
• the NOVl 8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 18A.
• NOVl 8a MEIL.YHVLFLVLECPN KKPP HMLSAMTVCSGGGVFLITGGIIYDVIVEPPSVG CG104251-01 SMTDEHGHQRPVAFFAYRV GQYIMEGLASSFLFT GG GFIILDQ NAPNIPKLNRF L FI GFVCVLi S I FMARVFMR KLPS YL.MG Protein Sequence Further analysis of the NOVl 8a protein yielded the following properties snown in Table 18B.
• NOVl 8a protein was found to have homology to the proteins shown in the BLASTP data in Table 18D.
• the NOVl 9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 19A.
• NOVl 9a protein was found to have homology to the proteins shown in the BLASTP data in Table 19D.
• NOV20 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 20A. Table 20A. NO 20 Sequence Analysis
• NOV20a protein Further analysis of the NOV20a protein yielded the following properties shown in Table 20C.
• NOV20a protein was found to have homology to the proteins shown in the BLASTP data in Table 20E.
• the NOV21 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 21 A.
• PSort 0.7900 probability located in plasma membrane; 0.3000 probability located in analysis: Golgi body; 0.2000 probability located in endoplasmic reticulum (membrane); 0.1007 probability located in microbody (peroxisome)
• NOV2 la protein was found to have homology to the proteins shown in the BLASTP data in Table 2 ID. •
• the NOV22 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 22A.
• NOV22a protein was found to have homology to the proteins shown in the BLASTP data in Table 22D.
• the NOV23 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 23A.
• PSort j 0.6850 probability located in endoplasmic reticulum (membrane); 0.6400 analysis: J probability located in plasma membrane; 0.4600 probability located in Golgi body; 0.1000 probability located in endoplasmic reticulum (lumen)
• NOV23a protein was found to have homology to the proteins shown in the BLASTP data in Table 23D.
• the NOV24 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 24A.
• NOV24a protein was found to have homology to the proteins shown in the BLASTP data in Table 24E.
• the NOV25 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 25A.
• NOV25a protein was found to have homology to the proteins shown in the BLASTP data in Table 25D.
• the NOV26 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 26A.
• PSort 0.8524 probability located in mitochondrial inner membrane 0.6000 analysis: probability located in endoplasmic reticulum (membrane); 0.3000 probability located in microbody (peroxisome); 0.2622 probability located in mitochondrial matrix space
• NOV26a protein was found to have homology to the proteins shown in the BLASTP data in Table 26E.
• the NOV27 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 27A.

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