EP2257641A1 - Farp2 and stk25 and uses thereof - Google Patents

Abstract

Methods and compositions are provided for treating HDL-associated diseases by altering the expression or activity of FARP2 or STK25 or a homolog or ortholog thereof.

Description

FARP2 AND STK25 AND USES THEREOF

GOVERNMENT SUPPORT This invention was supported in part by a grant from the American Heart Association Grant AHA Number: 0725905T. The U.S. government may have certain rights in the invention.

FIELD OF THE INVENTION The present invention relates generally to methods of treating a coronary artery disease or atherosclerotic condition. In particular, the methods comprise inhibiting regulating the expression and/or activity of a Farp2 and Stk25 to increase high-density lipoprotein levels.

BACKGROUND OF THE INVENTION

Atherosclerosis is characterized by lipid accumulation, inflammatory response, cell death, and fibrosis in the arterial wall. Atherosclerotic disease is the leading cause of morbidity and mortality, particularly in the United States and in Western European countries. Many risk factors are implicated in the development of atherosclerosis, including those that are genetically controlled, such as family history, high plasma low-density lipoprotein (LDL) levels and low plasma high-density lipoprotein (HDL) levels, hypertension, diabetes, old age, male sex, and lifestyle factors such as smoking, consuming fatty and overly processed food, and physical inactivity. Because LDL cholesterol (LDL-C) is proatherogenic, statin drugs were developed to lower plasma LDL-C levels and reduce the risk of adverse cardiovascular events. Recent studies suggest that reducing LDL-C levels to below current guideline targets further inhibits atherogenesis and reduces adverse coronary events. Statin drugs have reduced new adverse cardiovascular events by one-third; although this is significant, it is clear that additional therapies are needed. Although there is evidence that increasing plasma HDL cholesterol (HDL-C) levels inhibits atherogenesis, there do not appear to be many effective HDL-raising drugs in existence. Accordingly, a need exists to identify agents that increase HDL levels.

SUMMARY OF THE INVENTION The present invention stems from the unexpected finding that Farp2 and

Stk25, and suitable homologs and orthologs thereof, regulate the level of high density lipoprotein levels in plasma. The existence of HdIq 14 is confirmed by carrying out a cross between strains that had identical Soatl genes, did not differ in the key amino acid that defines the Λpoa2 QTL, but did differ in haplotype at the Hdlql4 region. When this cross confirmed the existence of HdIq 14, the QTL region was narrowed by combining crosses, comparative genomics, haplotype analysis, and expression and sequencing studies.

Thus the present invention is directed to the novel finding that Farp2 and Stk25, and suitable homologs and orthologs thereof, regulates the level of high density lipoprotein levels in plasma. The role of this protein in affecting plasma lipoprotein levels was previously unknown. This invention shows that Farp2 and Stk25 affect plasma levels of HDL cholesterol; specifically, Farp2 activity regulation and Stk25 activity inhibition can be used to raise plasma HDL cholesterol levels, and treat atherosclerotic disease. Accordingly, in one aspect, the invention pertains to use of an isolated antibody or functional fragment thereof, comprising an antigen-binding region that is specific for an epitope of a polypeptide encoded by a Farp gene (e.g., Farp2 gene ) or Stk gene (e.g., Stk25 gene), wherein the antibody or functional fragment binds to a surface receptor on a cell, and prevents or ameliorates development of a HDL- associated disease.

In one embodiment, the invention pertains to a method for treating a HDL- associated disease comprising administering to a subject an effective amount of the antibody or functional fragment thereof.

The antibodies of the invention can be formulated into a pharmaceutical composition comprising an antibody or functional fragment and a pharmaceutically acceptable carrier or excipient therefore. The pharmaceutical composition can be used as a method for treating a HDL-associated disease by administering to a subject in need thereof an effective amount of the pharmaceutical composition comprising the antibody or functional fragment.

The invention also pertains to use of an isolated antibody or functional fragment thereof for the preparation of a medicament for the treatment of a HDL- associated disease, wherein the antibody or functional fragment comprising an antigen-binding region that is specific for an epitope of a polypeptide encoded by a Farp gene or an Stk gene, or ortholog or homolog thereof (e.g., a Farp2 or Stk25 gene, or ortholog or homolog thereof). In one embodiment, the invention is directed to a method for treating a coronary artery disease or atherosclerotic condition comprising regulating the expression of a Farp or Stk. In a particular embodiment, the Farp is a Farp2 gene and the Stk is an Stk25 gene. In another embodiment, the step of inhibiting the expression and/or activity of a Farp or Stk further comprises inhibiting the activity using an isolated antibody or functional fragment thereof comprising an antigen-binding region that is specific for an epitope of a polypeptide encoded by a Farp gene (e.g., a Farp2 gene) or an Stk gene (e.g., an Stk25 gene), or homologs or orthologs thereof. In particular, the isolated antibody or functional fragment thereof comprising an antigen- binding region binds to a surface receptor on a cell and prevents or ameliorates the development of a HDL-associated disease. Also within the scope of the invention are transgenic animals carrying a gene encoding an antibody or functional fragment.

In one embodiment, the present invention is directed to a method for treating a coronary artery disease or atherosclerotic condition comprising altering the expression or activity of Farp2 or Stk25 or a homolog or ortholog thereof. In another embodiment, the present invention is directed to a method for detecting a coronary artery disease or susceptibility to a coronary artery disease comprising detecting an allele of Farp2 or Stk25 or a homolog or ortholog thereof that is indicative of a coronary artery disease or atherosclerotic condition. In a particular embodiment, the allele is Leu821 or Pro821 of FARP2. In another embodiment, the present invention is directed to a method for determining the efficacy of treating a coronary artery disease or atherosclerotic condition comprising treatment of a coronary artery disease or an atherosclerotic condition, and comparing the level oiFarp2 or Stk25 or a homolog or ortholog thereof with a reference such that the efficacy of treating the coronary artery disease or atherosclerotic condition is determined. In another embodiment, the present invention is directed to a method of identifying an agent useful for treating a coronary artery disease or an atherosclerotic condition, wherein altering the expression or activity of Farp2 or Stk25 or a homolog or ortholog thereof induces increased plasma HDL-C levels, comprising contacting a biological sample with a candidate agent and determining the level of total plasma lipoprotein or HDL-C in the sample before and after contact with the candidate agent, wherein an increase in HDL-C is indicative of an agent that is useful for treating a coronary artery disease or an atherosclerotic condition.

In another embodiment, the present invention is directed to a method for identifying an agent useful for treating a coronary artery disease or atherosclerotic condition comprising contacting FARP2 or STK25 or a homolog or ortholog thereof with a candidate agent in the presence of a known substrate, wherein an altered activity of the FARP2 or STK25 or a homolog or ortholog thereof identifies the candidate agent as an agent useful for treating a coronary artery disease or an atherosclerotic condition. In a particular embodiment, the contacting step is performed in a cultured cell. In another embodiment, the contacting step is performed in vivo. In a particular embodiment, the FARP2 or homolog or ortholog thereof is endogenous or exogenous.

In another embodiment, the present invention is directed to a method for modulating an HDL-associated disease comprising administering a modulating agent that elevates HDL-C levels in a subject. In a particular embodiment, the HDL- associated disease is selected from the group consisting of atherosclerosis, lipid disorders, Alzheimer's disease, oxidative stress, obesity, cardiovascular disease, type II diabetes and insulin resistance. In another embodiment, the lipid disorder is selected from the group consisting of: elevated cholesterol, dyslipidemic syndrome, elevated triglycerides, dyslipidemia, dyslipoproteinemia, hyperlipidemia, familial hypercholesterolemia, and familial hypertriglyceridemia. In a particular embodiment, the agent alters the expression or activity of FARP2 or STK25 or a homolog or ortholog thereof. In a particular embodiment, the modulating agent is selected from the group consisting of a small molecule, an antisense oligonucleotide, siRNA and an antibody. In a particular embodiment, the HDL-associated disease is any disease in which the HDL-C levels in the subject is below the accepted normal HDL-C level. In a particular embodiment, the HDL-associated disease is any disease in which the HDL-C levels in the subject is below the average HDL-C level of the related population. In a particular embodiment, the agent is administered with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Chr.l map of mouse HDL QTLs and their human concordant QTLs. A vertical line represents the chromosome, with the centromere at the top. Mouse HDL QTLs are represented by bars to the left of the chromosome. Each bar represents a QTL from one cross. QTL sizes are given either as 95% confidence interval (CI), 1.5-LOD drop intervals (if 95% CI are not available), or ± 10 cM centered around the LOD score peak (when neither CI nor LOD score figures are available). Mouse homologous regions of human HDL QTLs are represented by bars to the right of the chromosome, and the chromosome numbers of these human QTLs are to their right.

FIG. 2 shows HdIqU is confirmed in the NZB x NZW intercross. (A) Distribution of log transformed plasma HDL concentrations of 264 male and female F2 progeny. (B) Detailed LOD score plots for plasma HDL on Chr 1 from cross NZB x NZW (black line) and HdIqJ 4 is reduced by combining crosses (blue line). The X-axis depicts the marker positions in centimorgans, and the Y-axis depicts LOD scores. In combining crosses, data for Hdlql4 from both the present (NZB x NZW) F2 intercross and the previous (B6 ^χ 129) F2 intercross were combined using a statistical method to narrow the QTL. The second peak at cM 92 on blue line represents the QTL caused by the Apoa2 gene difference between B6 and 129. (C) The allele effect at peak marker D1MU336 on plasma HDL. WW, BB, and BW designate F2 mice homozygous for the NZW allele, NZB allele and heterozygous at the marker locus, respectively. Values are expressed as mean HDL ± SEM of F2 mice with a particular genotype at the designated locus. (P= 0.008 and 0.0002 in males and females, respectively).

FIG. 3 shows the Hdlql4 region narrowed by different strategies. (A) The 95% C/ is between Mb 82 to 162 in the B6xl29 cross. (B) The 95% C/ is between Mb 80 to 125 in the NZBxNZW cross. (C) Hdlql4 was reduced to a 125-gene 27.3- Mb region (Mb 89.8 to 117.1) by combining crosses. (D) Using crosses NZBxNZW and B6xl29 that detected Hdlql4, haplotyping reduced the region to 2.3 Mb containing 19 genes. (E) Using crosses B6xC3H and NONxNZO, which failed to detect Hdlql4, haplotyping further reduced the QTL to 2 genes.

FIG. 4 is a fine haplotyping map of the region ranging from Mb 89.8 to 96 on Chr 1 among stains 129, B6, NZB, and NZW. The physical position column indicates the distance, in megabases, from the centromere according to the Ensembl database (NCBI build 36). The mouse strains are indicated at the top of the figure. The SNPs are shown as observed nucleotides, A, T, G, and C, at each position among the strains. Lowercase SNP values are predicted based on surrounding haplotype. The genomic regions shared among strains 129 and NZW are highlighted green and that differed from regions shared by strains B6 and 129 are highlighted yellow. The common haplotype block was defined as to be three or more conservative shared alleles; common SNPs are shown as an outlined box according to the co-inheritance hypothesis.

FIG. 5 shows sequence comparisons. (A) The polymorphism causing Pro821Leu was identified by sequencing in 16 strains. The allele C (upper part) was identified in strains B6, C3H, CAST, PERA, NON, NZB, NZO, RIII, and SM, the allele T (lower part ) was identified in strains 129, A/J, DBA2, FVB, 1/LnJ, NOD, NZW, and SJL. (B) Proline 821 in FARP2 is in a conserved region among mammalian species.

FIG. 6 shows position and sequence data. (A) LOD score plots on Chr 1 in crosses NZB x NZW (black), NZO * NON (orange), B6 x C3H (red), and Pera x DBA (blue). The crosses, NON x NZO and B6 x C3H, do not have HdIq 14, the cross PERA x DBA2 has a QTL at that location. (B) Haplotype map for region harboring genes E030010N08Rik, Snedl, Mterfd2, Pask, Farp2, and Stk25 between strain pairs NZO and NON, B6 and C3H, and PERA and DBA/2. The haplotypes in crosses NON x NZO and B6 x C3H are identical for the bottom 5 SNPs, which includes the Cn SNP (95.4480 Mb) in Farp2 and all of the SNPs in Stk25. All of the SNPs above the bottom 5, however, are different. The cross PERA x DBA2 differs at the bottom 5 SNPs.

FIG. 7 shows HDL concentrations in inbred mouse strains with FARP2 variants Leu821 and Pro821 on background APO A2 Ala61 (top panel) and Valόl (lower panel), respectively. Top Panel: The 30 strains were separated into two types (12 strains with FARP2 Leu821 and 18 strains with FARP2 Pro821). Lower Panel: The 11 strains were separated into two types (6 strains with FARP2 Leu821 and 5 strains with FARP2 Pro821). Plasma HDL concentrations (mean ± SEM, mg/dL) were obtained from groups of 7- to 10-week-old females (left two columns) and males (right two columns) fasted for 4 h (Mouse Phenome Database). Each group consisted of between 10 and 40 mice (24 ± 7 males and 22 ± 6 females, mean ± SD).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected finding that HdIq 14 on

Chromosome 1 and that Farp2 and Stk25 are the likely underlying genes for the Quantitative Trait Locus (QTL) Hdlql4. Upon further examination, the FARP2

Leu⁸²¹ variant had significantly higher plasma HDL levels than those with the FARP2

Pro⁸²¹ variant.

Cardiovascular disease is the leading cause of morbidity and mortality in the

United States. Approximately 8% of CVD occurs in Americans under 50 years of age. It is estimated that approximately 40% of patients with premature CVD have low high-density lipoprotein cholesterol (HDL), and this represents the most common lipoprotein disorder in patients with CVD (Genest, J. et al, Circulation, 85:2025-

2033, 1992). This inverse relationship between HDL levels and CVD has generated interest in genetic factors contributing to variations in HDL levels. Because of the inherent difficulties of carrying out linkage analysis for HDL in humans, many mammalian geneticists have turned to the mouse model. The use of inbred strains of mice in this setting has proven to be a viable alternative to human genetic studies given the degree of control that can be exercised over experimental parameters such as environment, breeding scheme, and detailed phenotyping (Allayee, H. et al, Front. Biosci., 11 :1216-1226, 2006). More than 100 QTLs contributing to plasma HDL levels have been identified in the mouse, the most frequent one is identified on distal chromosome 1 , which is due to genetic variation in Apoa2 in each of the crosses. Disclosed herein is the confirmation of another QTL on proximal chromosome 1 for plasma HDL levels by controlling for the major gene effect of Apoa2 in NZB x NZW intercross. The peak of this locus is at D1MU336 (58.7 cM, 98.4Mb), and the 95% confidence interval is from 80 Mb through 125 Mb overlapped with Hdlql4 identified previously in cross B6 x 129. Syntenic analysis between mouse and human has indicated that part of the chromosomal region spanned by Hdlql4 in the mouse is homologous to the chromosomal region of 2q37.2-37.3 in humans. In humans, plasma HDL has been linked to chromosome 2q37.2-37.3 (Elbein, S. & Hasstedt, S., Diabetes, 51 :528-535, 2002), thus, it seems likely that the gene underlying HdIq 14 probably affects the HDL levels variation in both mouse and human, this assumption has been found in other traits, such as atherosclerosis (Wang, X. et al, Nature Gen., 37:365-372, 2005), body fat (Krade, H. et al, Nature Gen., 19:155-157, 1998), liver fibrogenesis (Hillebrandt, S. et al, Nature Gen., 37:835-843, 2005), blood pressure(Stoll, M. et al, Genome Res., 10:473-482, 2000; Sugiyama, F. et al, Genomics, 11 :10-77, 2001).

QTL identified from intercrosses often have large confidence intervals because they were detected from limited recombination events (Korstanje, R. & Paigen, B., Nature Gen., 31 :235-236, 2002). Several breeding strategies can be used to finely resolve QTL (Darvasi, A. , Nature Gen. , 18:19-24, 1998; McPeek, M. , Proc. Natl. Acad. Sci. USA, 97:12389-12390, 2000; Flint, J. et al, Nature Rev., 6:271-286, 2005), such as selective genotyping, recombinant progeny testing, interval-specific congenic lines, advanced intercross lines(Darvasi, A. & Soller, M., Genetics, 141 :1199-1207, 1995), recombinant inbred segregation test, recombinant inbred intercross test, and genetically heterogeneous stocks (Mott, R. et al, Proc. Natl. Acad. Sci. USA, 97: 12649-12654; 2000; Talbot, C. et al, Nature Gen., 21, 305-308, 1999). Bioinformatics tools are a rapid and economical approach for narrowing QTL (DiPetrillo, K. et al, Trends Genet, 21:683-692, 2005), which has been made possible by the recent investment in public sequence, genotype and expression databases, and the new development of statistics method. These tools including combining crosses, comparative genomics, and haplotype analysis, allow a stepwise narrowing of a QTL mapping interval, prioritizing candidate genes for further analysis with the potential of identifying the most probable candidate gene. By using this bioinformatics approach, the chromosomal region from 45 Mb spanned by the Hdlql4 in cross NZB x NZW was effectively reduced to 2.3 Mb, and the number of positional candidates was reduced from 225 to a testable 19. In the second step, we used the haplotypes of the parental strains in the 2 crosses that failed to detect a QTL, the NZOxNON and the B6xC3H crosses. Because the manuscript for these 2 crosses is still in preparation (Su, Paigen), the raw genotype and phenotype data has been archived in http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=projects/qtlprojlist. For these 2 crosses, we searched for a haplotype that is the same for all 4 strains because no QTL was detected. These crosses had identical haplotypes for the SNPs in the Farp2 and Stk25 genes but different haplotypes for the other 17 genes. Thus, these 2 crosses reduced the QTL region to just 2 genes and eliminated the other 17 genes (Figure 3, step e).

The positional candidate genes of HdIqH were further prioritized by gene sequencing and expression analysis. A QTL results from a difference between parental strains in the quantity of a protein or the function of protein. Therefore, identifying sequence polymorphisms between strains used to detect a QTL is important for determining the causal gene. A functional difference in the protein will result from a sequence change in the coding region of DNA, although the functional consequences are not always apparent. The complete coding sequences of these 15 positional candidate genes derived from B6, 129, NZB, and NZW, were compared and only six genes Glrpl, E030010N08Rik, Snedl, Mterfd2, Pask, and Farp2 were found that had polymorphisms changing amino acids. All the positional candidate genes were investigated by their expression difference of between strains 129, B6, and NZB that caused HdIq 14 QTL. Stk25 was found to have a significant expression difference either between parental strains B6 and 129 or between strains 129 and NZB, which have the different allele for Hdlql4, and no difference in expression between B6 and NZB, which both have the same allele for HdIq 14. This evaluation of expression differences shows that Stk25 is a viable candidate gene. Farp2 does have some expression difference, which is not entirely consistent across the strains, and also had a non-synonymous coding region difference (Leu821Pro) in a conserved region. The haplotype data shows that NZW, and 129 should have the same regulatory information as A/J at the Stk25, but differ from that in strains B 6 and NZB. The eQTL study in intercross between strains A/J and B6 showed that Stk25 was regulated by its eQTL, and the HDL levels is significant higher in mice with A/J homozygotes than that in mice with B6 homozygotes (52.1 ± 8.9 vs. 44.8 ± 7.9, p = 8.8 x 10^"6) in intercross derived from strains B6 and A/J, so strains A/J, 129, and NZW contribute the same high allele to HDL levels in different cross. Thus, it is likely that the expression of Stk25 is regulated by itself between strains 129 and B6, NZW and NZB. Stk25 encodes serine/threonine kinase 25, the serine/threonine kinase is a crucial regulator of AMPK activation in muscle and liver cells and (Imai, K. et al. , Biochem. Biophys. Res. Commun., 351 :595-601, 2006), therefore, its activity is of importance to our understanding of lipid metabolism. Although the variants changing amino acids were identified in genes Glrpl,

E030010N08Rik, Snedl, Mterfd2, and Pask, the HDL QTL identified in crosses NZO x NOD, B6 x C3H, and Pera x DBA were not likely due to the polymorphisms in these genes. Polymorphism Pro821 Leu of FARP2 are in a conserved pleckstrin homology (PH) domain (Lemmon, M., Biochem. Soc. Symp., 81-93, 2007), which itself is thought to bind lipids (Klopfenstein, D. & Vale, R., MoL Biol Cell, 15:3729- 3739, 2004). The mouse strains with the Leu821 variant were found to have significantly higher plasma HDL levels than those with Pro821 variant. These findings support Farp2 as the underlying HdIq 14 gene and suggest the Farp2 polymorphisms responsible for the HdIq 14 phenotype. Farp2 and Stk25 are very close to each other at the physical position, it is difficult to separate them using traditional genetic strategies such as congenic mice, and it is also unlikely to separate them by finding crossover in another cross because of the same haplotype among all the inbred strains.

Definitions As used herein, the term "HDL" refers to high density lipoprotein. HDL comprises a complex of lipids and proteins in approximately equal amounts that functions as a transporter of cholesterol in the blood. HDL is mainly synthesized in and secreted from the liver and epithelial cells of the small intestine. Immediately after secretion, HDL is in a form of a discoidal particle containing apoprotein A-I (also called apoA-I) and phospholipid as its major constituents, and also called nascent HDL. This nascent HDL receives, in blood, free cholesterol from cell membranes of peripheral cells or produced in the hydrolysis course of other lipoproteins, and forms mature spherical HDL while holding, at its hydrophobic center, cholesterol ester converted from said cholesterol by the action of LCAT (lecithin cholesterol acyltransferase). HDL plays an extremely important role in physiological function in terms of lipid metabolism called "reverse cholesterol transport system," which takes, in blood, excessive cholesterol out of peripheral tissues and transports it to the liver. High levels of HDL are associated with a decreased risk of atherosclerosis and coronary heart disease (CHD) as the reverse cholesterol transport system is considered to cause a prophylactic action on arteriosclerosis.

As used herein, the term "HDL modulating agent" refers to a any molecule able to alter the expression or functional levels of HDL such that the alteration in the HDL levels alters a HDL associated disease or condition. The HDL modulating agent can act directly or indirectly with a nucleic acid or polypeptide that effects HDL levels. Examples of HDL modulating agents include, but are not limited to antibodies, siRNA molecules, and low molecular weight compounds. In one embodiment, the HDL modulating agent is an isolated antibody or functional fragment thereof, comprising an antigen-binding region that is specific for an epitope of a polypeptide encoded by a Farp gene (e.g., a Farp2 gene) or Stk gene (e.g., a

Stk25 gene), or homologs or orthologs thereof. The antibody or functional fragment can bind to a surface receptor on a cell, and prevent or ameliorate development of a HDL-associated disease.

As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A "biological sample" further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. Most often, the sample has been removed from an animal, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, e.g., without removal from animal. Typically, a "biological sample" will contain cells from the animal, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, which can be used to measure the cancer-associated polynucleotide or polypeptides levels. A "biological sample" further refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as, for example, proteins or nucleic acid molecules. As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses a nucleic acid containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acids, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-0- methyl ribonucleotides, peptide-nucleic acids (PNAs). A nucleic acid sequence also encompasses naturally-occurring allelic variants of said nucleic acid. As used herein, the term "oligonucleotide" refers to a nucleic acid molecule consisting of two or more deoxyribonucleotides or ribonucleotides joined by phosphodiester bonds, and preferably containing between about 6 and about 300 nucleotides in length. The size of the oligonucleotide will depend on many factors, including the ultimate function or use of the oligonucleotide. Preferably, an oligonucleotide that functions, for example, as an extension primer will be sufficiently long to prime the synthesis of extension products in the presence of a catalyst, e.g. , DNA polymerase, and deoxynucleotide triphosphates. As used herein, the term "oligonucleotide" further refers to an oligonucleotide that has been modified structurally ("modified oligonucleotide") but functions similarly to the unmodified oligonucleotide. A modified oligonucleotide can contain non-naturally occurring portions, such as altered sugar moieties or inter- sugar linkages, such as a phosphorothioate.

As used herein, the term "polypeptide" refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. It also refers to either a full-length naturally-occurring amino acid sequence or a fragment thereof between about 8 and about 500 amino acids in length. Additionally, unnatural amino acids, for example, beta-alanine, phenyl glycine and homoarginine can be included. All of the amino acids used in the present invention can be either the D- or L-optical isomer. A polypeptide sequence also encompasses naturally- occurring allelic variants of said polypeptide. As used herein, "Quantitative trait locus" (QTL) analysis refers to a means of finding novel genes that regulate complex traits (Abiola, O. et al, Nature Rev., 4:91 1- 916, 2003). QTL analysis is particularly important for biomedical research because QTL detected in mouse models of disease often predict the location of human disease QTL. This location of disease QTL in homologous regions for both mice and humans suggests that the same genes regulate these traits in both species. Thus, QTL analyses using mice models can potentially identify genes that are important in human disease. As used herein, the term "subject" refers to an animal. Preferably, the animal is a mammal, either human or non-human. Also preferably, a subject refers to for example, primates (e.g. , monkeys, apes and humans), cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In a preferred embodiment, the subject is a human. As used herein, the term "efficacy" refers to the degree to which a desired effect is obtained. Specifically, the term refers to the degree to which plasma lipoprotein and HDL levels are modulated (e.g., elevated, increased, inhibited, reduced, or delayed). The term "efficacy" as used in the context of the present invention, also refers to relief or reduction of one or more symptoms or clinical events of a coronary artery disease (CAD). Relief or reduction of the symptoms include but are not limited to, a reduction or elimination of phosphatidylcholine degradation, oxidized phospholipids, a reduction in atherosclerotic plaque formation and rupture, a reduction in clinical events such as heart attack, angina, or stroke, a decrease in hypertension, a decrease in inflammatory mediator biosynthesis, reduction in plasma cholesterol, and the like. Relief or reduction of the symptoms can also refer to improving blood flow to vascular beds affected by atherosclerosis.

As used herein, the term "coronary artery disease" (CAD) or interchangeably "coronary heart disease" (CHD), refers to a cardiovascular disease characterized by blockage of the coronary arteries. Blockage can occur suddenly, by mechanisms such as plaque rupture or embolization. Blockage can occur progressively, with narrowing of the artery via myointimal hyperplasia and plaque formation. As a plaque thickens, the artery narrows and blood flow decreases, which results in a decrease in oxygen to the myocardium. This decrease in blood flow precipitates a series of consequences for the myocardium. For example, interruption in blood flow to the myocardium results in an "infarct" (myocardial infarction), which is commonly known as a heart attack. Those clinical signs and symptoms resulting from the blockage of arteries serving the heart are manifestations of CAD. Manifestations of CAD include angina, ischemia, myocardial infarction, cardiomyopathy, congestive heart failure, arrhythmias and aneurysm formation. It is understood that fragile plaque disease in the coronary circulation is associated with arterial thrombosis or distal embolization that manifests itself as a myocardial infarction. The CAD can cover a spectrum of disease stages. The early stage of the CAD is characterized with atheromatous streaks with the walls of the coronary arteries that do not obstruct the flow of blood. Over a period of years, these streaks increase in thickness. Thus, the next stage of CAD is characterized by the formation of plaques that expand into the walls of the arteries and the lumen of the vessel and affect the blood flow through the arteries. As the plaques grow in thickness and obstruct the majority of the diameter of the vessel, the subject is said to have developed symptoms of obstructive CAD or ischemic heart disease. The symptoms often include exertional angina or decreased exercise tolerance. As the degree of the CAD progresses, there can be near-complete obstruction of the lumen of the coronary artery, severely restricting the flow of oxygen-carrying blood to the myocardium. This stage of CAD is called myocardial infarction (heart attack), and is characterized with signs and symptoms of chronic coronary ischemia, including symptoms of angina at rest and flash pulmonary edema. As used herein, the term "atherosclerosis" refers to a process that leads to abnormal accumulation of cholesterol and cholesteryl esters and related lipids in macrophages, smooth muscle cell and other types of cells leading to narrowing and/or occlusion of one or several arteries and arterioles of the body and bodily organs, including but not limited to, the coronary arteries, aorta, renal arteries, corotid arteries, and arteries supplying blood to the limbs and central nervous system. The atherosclerosis can be quite insidious lasting for decades until atherosclerotic lesion, through physical forces from blood flow, becomes disrupted and deep arterial wall components are exposed to flowing blood, leading to thrombosis and compromised oxygen supply to target organs such as heart or brain. According to one theory, atherosclerosis involves the following stages: 1) endothelial cell dysfunction and/or injury, 2) monocyte recruitment and macrophage formation, 3) lipid deposition and modification, 4) vascular smooth muscle cell proliferation, and 5) synthesis of extracellular matrix. In addition, the associated inflammatory reactions and mediators of this pathologic process also are included in this definition. As used herein, "HDL- associated" disease refers to any disease or trait with low HDL levels, e.g., atherosclerosis. These associated diseases can include, for example, atherosclerosis, lipid disorders, Alzheimer's disease, oxidative stress, obesity, cardiovascular disease, type II diabetes and insulin resistance (also referred to as the metabolic syndrome). Lipid disorders can include, for example, elevated cholesterol (LDL levels of more than 130 milligrams per deciliter, or mg/dL),

Dyslipidemic syndrome, elevated triglycerides (triglyceride level as high as 1,500 mg/dL), dyslipidemia or dyslipoproteinemia (HDL is less than 35 mg/dL), hyperlipidemia or high cholesterol, familial hypercholesterolemia (a genetic disorder that increases total and LDL cholesterol), and familial hypertriglyceridemia (inherited high triglycerides). As used herein, the term "a significant change in the expression level" refers to either an increase or a decrease of the expression level from the control level by an amount greater than the standard error of the assay employed to assess expression. The term also refers to a change by preferably at least about 10%, about 20%, about 25%, about 30%, preferably at least about 40%, about 50%, more preferably at least about 60%, about 70%, or about 90%, about 100%, about 150%, or about 200%, or greater.

As used herein, the term "gene" refers to a nucleic acid sequence that encodes and regulates expression of a polypeptide. A gene includes, therefore, regulatory elements, e.g., promoters, splice sites, enhancers, repressor binding sites, etc. A gene can have many different "alleles," which are sequence variations that can affect the polypeptide sequence or expression level, or have no effect on the polypeptide. A gene can include one or more "open reading frames," which are nucleic acid sequences that encode a contiguous polypeptide. A gene can be present either endogenously or exogenously. As used herein, the term "expression level" (e.g., of Farp2 or Stk25) refers to the amount of mRNA transcribed from the corresponding gene that is present in a biological sample. The expression level can be detected with or without comparison to a level from a control sample or a level expected of a control sample.

As used herein, the term "control level" refers to a standard level of a biomarker by which a change is measure against. In one embodiment, the "control level" can be a normal level of a biomarker nucleic acid expression, or a biomarker polypeptide, or a biomarker biological activity from normal or healthy cells, tissues, or subjects, or from a population of normal or healthy cells, tissues, or subjects. By way of non-limiting example, a control level can be levels of Farp2 or Stk25 polypeptide or biological activity in a normal cell, tissue, or subjects, or plasma total lipoprotein or HDL levels. As used herein, the term "control expression level" (e.g., of Farp2 or Stk25) refers to the amount of mRNA transcribed from the corresponding gene that is present in a biological sample representative of healthy subjects. The term "control expression level" can also refer to an established level of mRNA representative of the healthy population that has been previously established based on measurement from healthy subjects.

As used herein, "detecting" refers to the identification of the presence or absence of a molecule in a sample. Where the molecule to be detected is a polypeptide, the step of detecting can be performed, for example, by binding the polypeptide to an antibody that is detectably labeled. A detectable label is a molecule that is capable of generating, either independently, or in response to a stimulus, an observable signal. A detectable label can be, but is not limited to a fluorescent label, a chromogenic label, a luminescent label, or a radioactive label. Methods for "detecting" a label include, for example, quantitative and qualitative methods adapted for standard or confocal microscopy, FACS analysis, and those adapted for high throughput methods involving multiwell plates, arrays or microarrays. One of ordinary skill in the art can select appropriate filter sets and excitation energy sources for the detection of fluorescent emission from a given fluorescent polypeptide or dye. "Detecting" as used herein can also include the use of multiple antibodies to a polypeptide to be detected, wherein the multiple antibodies bind to different epitopes on the polypeptide to be detected. Antibodies used in this manner can employ two or more detectable labels, and can include, for example a FRET pair. A polypeptide molecule is "detected" according to the present invention when the level of detectable signal is at all greater than the background level of the detectable label, or where the level of measured polypeptide is at all greater than the level measured in a control sample.

As used herein, "detecting" also refers to identification of the presence of a target nucleic acid molecule, for example, by a process wherein the signal generated by a directly or indirectly labeled probe nucleic acid molecule (capable of hybridizing to a target in a serum sample) is measured or observed. Detection of the probe nucleic acid is directly indicative of the presence, and thus the detection, of a target nucleic acid, such as a sequence encoding a marker gene. Methods and techniques for "detecting" fluorescent, radioactive, and other chemical labels may be found in Ausubel et al. (1995, Short Protocols in Molecular Biology, 3rd Ed. John Wiley and Sons, Inc.). Alternatively, a nucleic acid can be "indirectly detected" wherein a moiety is attached to a probe nucleic acid that will hybridize with the target, wherein the moiety comprises, for example, an enzyme activity, allowing detection of the target in the presence of an appropriate substrate, or a specific antigen or other marker allowing detection by addition of an antibody or other specific indicator. Alternatively, a target nucleic acid molecule can be detected by amplifying a nucleic acid sample prepared from a patient clinical sample, using oligonucleotide primers that are specifically designed to hybridize with a portion of the target nucleic acid sequence. Quantitative amplification methods, such as, but not limited to TaqMan^®, can also be used to "detect" a target nucleic acid according to the invention. A nucleic acid molecule is "detected" as used herein where the level of nucleic acid measured (such as by quantitative PCR), or the level of detectable signal provided by the detectable label is at all above the background level.

As used herein, "detecting" further refers to at least the early detection of CADs such as atherosclerosis in a subject, wherein the "early" detection refers to the detection of CADs at an early stage, preferably, prior to a time when a symptom is visible. "Detecting" as used herein further refers to the detection of CADs recurrence in a subject, using the same detection criteria as indicated above. "Detecting" as used herein further refers to the measurement of a change in the degree of the CADs before and after treatment with a therapeutic compound. In this case, a change in degree of the CADs in response to a therapeutic compound refers to either an increase or a decrease by at least about 10% in the expression of one or more marker genes, or alternatively, in the amount of the marker gene polypeptides presented in a clinical sample, in response to the presence of a therapeutic compound relative to the expression level in the absence of the therapeutic compound. The term "antibody" as used herein refers to an intact antibody or an antigen binding fragment (i.e., "antigen-binding portion") or single chain (i.e., light or heavy chain) thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHl, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.

The term "antigen binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. Antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; an F(ab)2 fragment, a bivalent fragment comprising two Fab fragments (generally one from a heavy chain and one from a light chain) linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CHl domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al, 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies include one or more "antigen binding portions" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen binding portions can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v- NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). Antigen binding portions of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

Antigen binding portions can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10):1057-1062; and U.S. Pat. No. 5,641,870).

The terms "silence" and "inhibit the expression of, in as far as they refer to a target gene e.g. Farp2 or Stk25, herein refer to the at least partial suppression of the expression of the Farp2 or Stk25 gene, as manifested by a reduction of the amount of mRNA transcribed from the Farp2 or Stk25 gene which may be isolated from a first cell or group of cells in which the Farp2 or Stk25 gene is transcribed and which has or have been treated such that the expression of the Farp2 or Stk25 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

(mRNA in control cells) - (mRNA in treated cells) , . _Λn . — • 100%

(mRNA in control cells)

Alternatively, the degree of inhibition maybe given in terms of a reduction of a parameter that is functionally linked to the Farp2 or Stk25 gene transcription, e.g. the amount of protein encoded by the Farp2 or Stk25 gene which is secreted by a cell, or the number of cells displaying a certain phenotype. In principle, the Farp2 or Stk25 gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay.

As used herein, the term "a," "an," "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The present invention is based on the novel and surprising discovery that Farp2 and Stk25 regulate the level of plasma total lipoprotein and HDL, the levels of which correlate to the development and progression of CADs. In particular, alleles of Farp2 that alter the expression and activity of the gene product increases HDL levels. Accordingly, one aspect of the present invention provides methods for identifying a compound that alters the expression and/or activity of FARP2, STK25 and related genes and gene products (e.g., homologs and orthologs, e.g., human orthologs). Another aspect of the present invention provides uses of FARP2, STK25 and orthologs thereof as a biomarker for monitoring the onset, progression, or regression of a CAD, or for assessing the efficacy of a compound in treating a CAD.

Screening assays

In one aspect, the present invention provides a method for screening (identifying) a compound that alter the activity or expression of FARP2, STK25 and homologs and orthologs thereof, thereby increasing the plasma level of HDL-C. The screening can be performed, for example, by contacting a compound with a biological sample containing a FARP2 or STK25 gene product and monitoring the effect of the compound on the activity FARP2 or STK25 (or a suitable homolog or ortholog thereof), monitoring the expression of FARP2 or STK25 (or a suitable homolog or ortholog thereof), or monitoring the effect of the compound on lipoprotein and HDL- C levels in the sample. Farp2 or Stk25 (or a suitable homolog or ortholog thereof) can be in the form of an endogenous or exogenous nucleic acid molecule {e.g., endogenous gene or exogenous vector comprising a suitable reading frame), or a polypeptide, or functional fragments thereof. The effect of the compound on modulating the level of total plasma lipoprotein or HDL-C is via modulating the activity of, for example, FARP2 or STK25 (or a suitable homolog or ortholog thereof). The modulation of the activity can include, but is not limited to: 1) inhibiting or preventing the polypeptide or functional fragments thereof from degrading the HDL, 2) degrading or inducing degradation of the polypeptide or functional fragments thereof, 3) inactivating the biological activity of the polypeptide or the functional fragments thereof; 4) reducing or inhibiting the expression of the nucleic acid molecules; and 5) degrading or destabilizing nucleic acid molecules.

For purposes of determining the effects of the compound on the HDL, a parallel sample that does not receive the compound is also monitored as a control. The treated and untreated samples are then compared by any suitable phenotypic criteria, including but not limited to, microscopic analysis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide or the complex thereof, the level of enzymatic activity, and the ability of the cells to interact with other cells or compounds, etc. Differences between the treated and untreated cells indicate effects attributable to the compound. In one embodiment, the compound can be identified for inhibiting the activity or modulating the levels of total plasma lipoprotein or HDL-C by at least about 10%, about 20%, about 30%, about 50%, about 70%, about 90%, about 100%, about 150%, about 200% or more. The steps of the screening method include 1) contacting the compound with a first biological sample comprising HDL and a suitable source of FARP2 or STK25 (or a suitable homolog or ortholog thereof), determining level of the HDL in the first biological sample; 3) determining level of the HDL in a second biological sample wherein the second biological sample has not been exposed to the compound; and 4) selecting the compound wherein the level of the HDL from 2) is at least about 1.5 fold as the level of HDL from 3).

In one embodiment, the screening assay is a cell-free assay where a cell-free biological sample containing HDL and a suitable FARP2 or STK25 (or a suitable homolog or ortholog thereof), is contacted with a compound, and the ability of the compound to modulate total plasma lipoprotein or HDL-C levels is determined. Methods of measuring HDL levels are known in the art (Sugiuchi et al, CHn. Chem., 41 :717-723, 1995; Izawa et al., J. Med. Pharm. ScL, 37:1385-1388, 1997).

For the cell-free screening assay described herein, the suitable FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide or the functional fragments thereof can be contained in the biological sample itself, or added into the biological sample from other sources. For example, the polypeptide or the functional fragments thereof can be commercially available, or purified in significant amounts from an appropriate biological source, e.g. , cultured cells. Alternatively, the proteins can be recombinantly produced from an isolated gene or cDNA by expression in a suitable prokaryotic or eukaryotic expression system, and thereafter purified, as is also known in the art. Likewise, the HDL can be contained in the biological sample itself, or added into the biological sample from other sources. The HDL can be fully isolated or partially isolated. Methods of partially or completely isolating HDL are known to those of skill in the art (Havel et al, J. CHn. Invest., 43:1345-1353, 1955; Navab et al., J. CHn. Invest, 99:2005-2019, 1997; Carroll and Rudel, J. Lipid Res., 24:200-207, 1983, McNamara et al, CHn. Chem., 40:233-239, 1994, Grauholt et al, Scandinavian J. CHn. Lab. Invest, 46:715-721, 1986; Warnick et al, CHn. Chem., 28:1379-1388, 1982; Talameh et al, CHn. Chimica Acta, 158:33-41, 1986).

In another embodiment, the screening assay is an in vivo screening assay. The in vivo screening assay can be carried out in non-human animals to discover compounds that effectively inhibit, reduce, or delay degradation of HDL in the animals. In one non-limiting example, a compound is administered to a non-human animal, optionally following a high- fat diet, at a suitable dosage for a suitable amount of time. The animal is then bled, plasma lipoproteins are isolated, and the HDL level is determined by methods known in the art. An increase in the HDL level in the animal treated with the compound compared to the HDL level in the animal not treated with the compound, indicates that the compound inhibits the activity of, for example, FARP2 or STK25 (or a suitable homolog or ortholog thereof), thereby modulating the levels of total plasma lipoprotein and/or HDL-C in the animal. Preferably, the increase is at least about 1.5 fold. Also preferably, the compound modulates the level of total plasma lipoproteins and/or HDL-C in the animal by at least about 10%, about 20%, about 30%, about 50%, about 70%, about 90%, about 100%, about 150%, about 200% or more.

Optionally, prior to being administered to the animal, the compound can be pre-screened by the cell-free screening assay as described herein, or a cell-based screening assay. In the cell-based screening assay, a cell expressing a suitable FARP2 or

STK25 (or a suitable homolog or ortholog thereof), or functional fragment(s) thereof is contacted with a compound, and the ability of the compound to modulate the activity is determined. Determining the ability of the compound to modulate the activity can be accomplished by assessing the biological activity, such as catalytic/enzymatic activity of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) for an appropriate substrate, by assessing the ability of the compound to bind to or interact with the FARP2 or STK25 (or a suitable homolog or ortholog thereof), by assessing induction of a reporter gene comprising a responsive element operatively linked to a nucleic acid encoding a detectable marker, or by assessing a suitably regulated cellular response, for example, signal transduction or protein/protein interactions. The cell can be a mammalian cell, an insect cell, a bacterial cell, or a yeast cell, etc.

Another aspect of the present invention pertains to the compound obtained from the above screening assays. The compound can be a chemical compound, an antisense oligonucleotide, a siRNA, a non-immunoglobulin binding scaffold or an antibody. Antibodies

In one embodiment, the invention pertains to modulating the HDL nucleic acid and polypeptide levels by using antibodies. An antibody can include, but is not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single chain antibodies, Fab fragments, Fv fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-iodiotypic antibodies, or other epitope binding polypeptide. An antibody of the present invention can be monospecific, dispecfϊc, trispecific, or of greater multispecificity. Preferably, an antibody, useful in the present invention for the detection of the mouse ES 1 or human CESl polypeptide, is a human antibody or fragment thereof, including scFv, Fab, Fab', F(ab'), Fd, single chain antibody, of Fv. An antibody, useful in the invention can include a complete heavy or light chain constant region, or a portion thereof, or an absence thereof. In one embodiment, an antibody useful in the invention can be a humanized antibody, in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability. Methods for making humanized antibodies are known in the art (Teng et al, Proc. Natl. Acad. ScL USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3- 16, 1982; WO 92/06193; and EP 0239400).

In some embodiments, antigen binding portions of antibodies that bind to a Farp or Stk polypeptide, (e.g., VH andVL chains) can be "mixed and matched" to create other anti-Farp or Stk binding molecules. The binding of such "mixed and matched" antibodies can be tested using binding assays (e.g., ELISAs). When selecting a VH to mix and match with a particular VL sequence, typically one selects a VH that is structurally similar to the VH it replaces in the pairing with that VL. Likewise a full length heavy chain sequence from a particular full length heavy chain/full length light chain pairing is generally replaced with a structurally similar full length heavy chain sequence. Likewise, a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence. Likewise a full length light chain sequence from a particular full length heavy chain/full length light chain pairing should be replaced with a structurally similar full length light chain sequence. Identifying structural similarity in this context is a process well known in the art.

A human antibody comprises heavy or light chain variable regions or full length heavy or light chains that are "the product of or "derived from" a particular germline sequence if the variable regions or full length chains of the antibody are obtained from a system that uses human germline immunoglobulin genes as the source of the sequences. In one such system, a human antibody is raised in a transgenic mouse carrying human immunoglobulin genes. The transgenic is immunized with the antigen of interest (e.g., an epitope of a Farp or Stk polypeptide). A human antibody that is "the product of or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody. A human antibody that is "the product of or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline-encoded sequence, due to, for example, naturally occurring somatic mutations or artificial site-directed mutations. However, a selected human antibody typically has an amino acid sequence at least 90% identical to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.

Camelid antibodies

Antibody proteins obtained from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family, including New World members such as llama species (Lama paccos, Lama glama and Lama vicugna), have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies found in nature in this family of mammals lack light chains, and are thus structurally distinct from the four chain quaternary structure having two heavy and two light chains typical for antibodies from other animals. See WO 94/04678.

A region of the camelid antibody that is the small, single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight, antibody-derived protein known as a "camelid nanobody". See U.S. Pat. No. 5,759,808; see also Stijlemans et al., 2004 J. Biol. Chem. 279: 1256-1261; Dumoulin et al., 2003 Nature 424: 783-788; Pleschberger et al., 2003 Bioconjugate Chem. 14: 440-448; Cortez- Retamozo et al., 2002 hit. J. Cancer 89: 456-62; and Lauwereys. et al., 1998 EMBO J. 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be "humanized". Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus, yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

The low molecular weight and compact size further result in camelid nanobodies' being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitate drug transport across the blood brain barrier. See U.S. Pat. Pub. No. 20040161738, published August 19, 2004. These features combined with the low antigenicity in humans indicate great therapeutic potential. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli. Also included in the scope of the present invention are camelid antibodies. Accordingly, a feature of the present invention is a camelid antibody or camelid nanobody having high affinity for the Farp or Stk polypeptide.

Diabodies

Diabodies are bivalent, bispecific molecules in which VH and VL domains are expressed on a single polypeptide chain, connected by a linker that is too short to allow for pairing between the two domains on the same chain. The VH and VL domains pair with complementary domains of another chain, thereby creating two antigen binding sites (see e.g., Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994 Structure 2:1121-1123). Diabodies can be produced by expressing two polypeptide chains with either the structure VHA-VLB and VHB- VLA (VH-VL configuration), or VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. Most of them can be expressed in soluble form in bacteria.

Single chain diabodies (scDb) are produced by connecting the two diabody- forming polypeptide chains with linker of approximately 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Irnrnunother., 45(3-4): 128-30; Wu et al., 1996 hnmunotechnology, 2(l):21-36). scDb can be expressed in bacteria in soluble, active monomelic form (see Holliger and Winter, 1997 Cancer Immunol.

Immunother., 45(34): 128-30; Wu et al., 1996 Immunotechnology, 2(l):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83-105; Ridgway et al., 1996 Protein Eng., 9(7):617-21). A diabody can be fused to Fc to generate a "di-diabody" (see Lu et al., 2004 J. Biol. Chem., 279(4):2856-65).

Engineered and modified antibodies An antibody of the invention can be prepared using an antibody having one or more VH and/or VL sequences as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i. e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody- antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., 1998 Nature 332:323-327; Jones et al., 1986 Nature 321 :522-525; Queen et al., 1989 Proc. Natl. Acad. See. U.S.A. 86:10029- 10033; U.S. Pat. No. 5,225,539, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc- cpe.cam.ac.uk/vbase), as well as in Kabat et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992 J. MoI. Biol. 227:776-798; and Cox et al., 1994 Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference. The VH CDRl, 2 and 3 sequences and the VL CDRl, 2 and 3 sequences can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

CDRs can also be grafted into framework regions of polypeptides other than immunoglobulin domains. Appropriate scaffolds form a conformationally stable framework that displays the grafted residues such that they form a localized surface and bind the target of interest. For example, CDRs can be grafted onto a scaffold in which the framework regions are based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CPl zinc finger, PSTl, coiled coil, LACI-Dl, Z domain or tendramisat (See e.g., Nygren and Uhlen, 1997 Current Opinion in Structural Biology, 7, 463-469).

Another type of variable region modification is mutation of amino acid residues within the VH and/or VL CDRl, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation." Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein. Conservative modifications can be introduced. The mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and/or VL, e.g., to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site- directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell -epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Pat. Pub. No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework or CDR regions, antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.

In one embodiment, the hinge region of CHl is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHl is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, U.S. Pat. No. 6,277,375 describes the following mutations in an IgG that increase its half-life in vivo: T252L, T254S, T256F. Alternatively, to increase the biological half life, the antibody can be altered within the CHl or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered CIq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. Nos. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in WO 94/29351 by Bodmer et al.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids. This approach is described further in WO 00/42072 by Presta. Moreover, the binding sites on human IgGl for FcγRl, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al, 2001 J. Biol. Chem. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Pub. WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, RX. et al., 2002 J. Biol. Chem. 277:26733-26740). WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein- modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG moieties become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl-ClO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

In addition, pegylation can be achieved in any part of an antibody by the introduction of a nonnatural amino acid. Certain nonnatural amino acids can be introduced by the technology described in Deiters et al., J Am Chem Soc 125: 11782- 11783, 2003; Wang and Schultz, Science 301:964-967, 2003; Wang et al., Science 292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in US Patent No. 7,083,970. Briefly, some of these expression systems involve site-directed mutagenesis to introduce a nonsense codon, such as an amber TAG, into the open reading frame encoding a polypeptide of the invention. Such expression vectors are then introduced into a host that can utilize a tRNA specific for the introduced nonsense codon and charged with the nonnatural amino acid of choice. Particular nonnatural amino acids that are beneficial for purpose of conjugating moieties to the polypeptides of the invention include those with acetylene and azido side chains. The polypeptides containing these novel amino acids can then be pegylated at these chosen sites in the protein. Methods of engineering antibodies

Antibodies can be modified to create new antibodies by modifying full length heavy chain and/or light chain sequences, VH and/or VL sequences, or the constant region(s) attached thereto. For example, one or more CDR regions of the antibodies can be combined recombinantly with known framework regions and/or other CDRs to create new, recombinantly-engineered antibodies. The starting material for the engineering method is one or more of the VH and/or VL sequences, or one or more CDR regions thereof. To create the engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VL sequences, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as the starting material to create a "second generation" sequence(s) derived from the original sequence(s) and then the "second generation" sequence(s) is prepared and expressed as a protein. Standard molecular biology techniques can be used to prepare and express the altered antibody sequence. The antibody encoded by the altered antibody sequence(s) is one that retains one, some or all of the desired functional properties from which it is derived. The functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein (e.g., ELISAs). In certain embodiments of the methods of engineering antibodies of the invention, mutations can be introduced randomly or selectively along all or part of an antibody coding sequence. For example, PCT Pub. WO 02/092780 by Short describes methods for creating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, WO 03/074679 by Lazar et al. describes methods of using computational screening methods to optimize physiochemical properties of antibodies.

Non-antibody binding molecules The invention further provides binding molecules that exhibit functional properties of antibodies but derive their framework and antigen binding portions from other polypeptides (e.g., polypeptides other than those encoded by antibody genes or generated by the recombination of antibody genes in vivo). The antigen binding domains of these binding molecules are generated through a directed evolution process. See U.S. Pat. No. 7,115,396. Molecules that have an overall fold similar to that of a variable domain of an antibody (an "immunoglobulin-like" fold) are appropriate scaffold proteins. Scaffold proteins suitable for deriving antigen binding molecules include fibronectin or a fibronectin dimer, tenascin, N-cadherin, E- cadherin, ICAM, titin, GCSF-receptor, cytokine receptor, glycosidase inhibitor, antibiotic chromoprotein, myelin membrane adhesion molecule PO, CD8, CD4, CD2, class I MHC, T-cell antigen receptor, CDl, C2 and I-set domains of VCAM-I, I-set immunoglobulin domain of myo sin-binding protein C, I-set immunoglobulin domain of myosin-binding protein H, I-set immunoglobulin domain of telokin, NCAM, twitchin, neuroglian, growth hormone receptor, erythropoietin receptor, prolactin receptor, interferon-gamma receptor, D-galactosidase/glucuronidase, D- glucuronidase, transglutaminase, T-cell antigen receptor, superoxide dismutase, tissue factor domain, cytochrome F, green fluorescent protein, GroEL, and thaumatin.

The antigen binding domain (e.g., the immunoglobulin-like fold) of the non- antibody binding molecule can have a molecular mass less than 10 kD or greater than 7.5 kD (e.g., a molecular mass between 7.5-10 kD). The protein used to derive the antigen binding domain is a naturally occurring mammalian protein (e.g., a human protein), and the antigen binding domain includes up to 50% (e.g., up to 34%, 25%, 20%, or 15%), mutated amino acids as compared to the immunoglobulin-like fold of the protein from which it is derived. The domain having the immunoglobulin-like fold generally consists of 50-150 amino acids (e.g., 40-60 amino acids). To generate non-antibody binding molecules, a library of clones is created in which sequences in regions of the scaffold protein that form antigen binding surfaces (e.g., regions analogous in position and structure to CDRs of an antibody variable domain immunoglobulin fold) are randomized. Library clones are tested for specific binding to the antigen of interest and for other functions. Selected clones can be used as the basis for further randomization and selection to produce derivatives of higher affinity for the antigen. High affinity binding molecules are generated, for example, using the tenth module of fibronectin III (10Fn3) as the scaffold. A library is constructed for each of three CDR-like loops of 10FN3 at residues 23-29, 52-55, and 78-87. To construct each library, DNA segments encoding sequence overlapping each CDR-like region are randomized by oligonucleotide synthesis. Techniques for producing selectable 10Fn3 libraries are described in U.S. Pat. Nos. 6,818,418 and 7,115,396; Roberts and Szostak, 1997 Proc. Natl. Acad. Sci USA 94:12297; U.S. Pat. No. 6,261,804; U.S. Pat. No. 6,258,558; and Szostak et al. WO98/31700.

Non-antibody binding molecules can be produced as dimers or multimers to increase avidity for the target antigen. For example, the antigen binding domain is expressed as a fusion with a constant region (Fc) of an antibody that forms Fc-Fc dimers. See, e.g., U.S. Pat. No. 7,115,396.

RNA Interference Antisense oligonucleotide refers to a polynucleotide that is complementary to all or part of a target primary transcript (unprocessed transcript) or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO 9928508.). The complementarity of an antisense oligonucleotide may be with any part of the specific gene transcript, e.g., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence.

A siRNA refers to a small interfering RNA, which acts to degrade mRNA sequences homologous to either of the RNA strands in the duplex and can cause post- transcriptional silencing of specific genes in cells, for example, mammalian cells (including human cells) and in the body, for example, mammalian bodies (including humans). The phenomenon of RNA interference is known in the art (Bass, Nature, 411 :428-29, 2001 ; Elbahir et al. , Nature, 411 :494- 98, 2001 ; Fire et al. , Nature, 391:806-11, 1998; and WO 01/75164). The siRNAs based upon the sequences and nucleic acids encoding the gene products disclosed herein typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches. The siRNAs are capable of causing interference and can cause post-transcriptional silencing of specific genes in cells, for example, mammalian cells (including human cells) and in the body, for example, mammalian bodies (including humans). Exemplary siRNAs according to the present invention can have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integer thereabout or there between. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.) and Ambion, Inc. (Austin, Tex.).

Double-stranded ribonucleic acid (dsRNA) molecules can also be used for inhibiting the expression of the target gene (e.g., Farp2 or Stk25) in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the target gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein the dsRNA, upon contact with a cell expressing the target gene, inhibits the expression of the target gene by at least 10%, 25%, or 40%. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the target gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single- stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21 , base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888).

In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2' modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of non-natural bases into the oligonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with alternate linkages such as thiophosphates. More than one such modification may be employed.

Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Generally, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2-chloroethyl)amine; N-acetyl-N'- (p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, DJ., and K.B. Hall, Biochem. (1996) 35:14665- 14670). In a particular embodiment, the 5'-end of the antisense strand and the 3'-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds.

In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the degradation activities of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the degradation activity of cellular enzymes against nucleic acids are known in the art including, but not limited to, 2'-amino modifications, 2 '-amino sugar modifications, 2'-F sugar modifications, 2'-F modifications, 2'-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2'-O- methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1 :1116-8). Thus, at least one 2'-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, generally by a 2'-amino or a 2'-methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2 '-oxygen of ribose with the 4' -carbon of ribose. Oligonucleotides containing the locked nucleotide are described in Koshkin, A.A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D.A. and D.R. Corey, Chem. Biol. (2001), 8:1-7).

Use ofFARP2, STK25 or a Homolog/Ortholog thereof As A Biomarker

In one aspect, the expression levels of the differentially expressed Farp2 or Stk25 (or a suitable homolog or ortholog thereof) genes are determined in normal and CAD cells and/or tissues. In one embodiment, the methods of determining the expression levels of the gene(s) can comprise one or more of the following steps in any effective order, e.g., contacting a biological sample with a polynucleotide probe under conditions effective for said probe to hybridize specifically to the FARP2 or STK25 (or a suitable homolog or ortholog thereof) nucleic acid molecule in said sample, and detecting the presence or absence of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) marker gene nucleic acid in said sample. Specific alleles, comprising distinct and relevant polymorphisms are also detected.

In one embodiment, the probe is applied to the samples obtained from both the normal and CAD cells and/or tissues, and the presence of the Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid molecule is detected with the methods known in the art. For example, the methods of detecting the presence of the marker genes can be carried out by any effective process, e.g., by Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, etc.

In another embodiment, the probe is applied to the samples obtained from both the normal and CAD cells and/or tissues, and the amount of the Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid is detected with the methods known in the art. Such methods can involve, e.g. , contacting with probe, hybridizing, and detecting hybridized probe, but using more quantitative methods and/or comparisons to standards. The amount of hybridization between the probe and target can be determined by any suitable methods, e.g. , PCR, RT-PCR, RACE PCR, Northern blot, polynucleotide microarrays, Rapid-Scan, etc., and includes both quantitative and qualitative measurements.

In another embodiment, FARP2 or STK25 (or a suitable homolog or ortholog thereof) specific antibodies can be used to detect the presence of FARP2 or STK25 (or a suitable homolog or ortholog thereof), or a fragment(s) thereof, in a biological sample by any method known in the art. The method can include immunoassays such as competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are known in the art (Ausubel et al. , eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). In addition, immunoassays useful in the present invention can also include both homogeneous and heterogeneous procedures such as fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), and nephelometric inhibition immunoassay (NIA).

In another embodiment, the level of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide in a biological sample can be determined as a way of monitoring the expression level of FARP2 or STK25 (or a suitable homolog or ortholog thereof). Such a method would include, for example, the steps of obtaining a biological sample, contacting the sample with an antibody specific for the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide or suitable epitope thereof, and determining the amount of immune complex formation with the antibody, with the amount of immune complex formation being indicative of the level of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide. This determination is instructive when the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide level in a biological sample obtained from a subject with a CAD is compared to the level of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) in a biological sample taken from a normal subject, or in one or more samples previously or subsequently obtained from the same subject.

Determination of the amount of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide can also be correlated with progression of a CAD. The FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide level can be used predictably to evaluate whether a biological sample contains cells that are predisposed towards becoming CADs, or can be used to plan a particular therapeutic regimen.

Diagnostic assays The determination of a detectable increase or decrease in the expression level of FARP2 or STK25 (or a suitable homolog or ortholog thereof) in a subject with a CAD as compared to a normal subject, provides a means of diagnosing or monitoring the disease status, and/or response to benefit to a therapy. Therefore, the present invention provides methods for detecting a CAD or atherosclerotic condition, or alternatively determining whether a subject is at risk for developing a CAD or atherosclerotic condition.

In clinical applications, human tissue samples can be screened for the presence and/or absence of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) encoding nucleic acid and/or FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptides. Such samples can comprise tissue samples, whole cells, cell lysates, or isolated nucleic acids, including, for example, needle biopsy cores, surgical resection samples, lymph node tissue, plasma, or serum. In certain embodiments, nucleic acids extracted from these samples may be amplified using techniques well known in the art. The levels FARP2 or STK25 (or a suitable homolog or ortholog thereof) and/or plasma lipoprotein or HDL-C detected would be compared with those in a normal tissue sample.

In one embodiment, the diagnostic method comprises determining whether a subject has a CAD by detecting the mRNA, cDNA or polypeptide level of Farp2 or Stk25 (or a suitable homolog or ortholog thereof). A significant change in the expression level of the activity in the subject compared to that in the normal healthy subject is an indication of a CAD or a susceptibility to a CAD. Preferably, the change is at least about 10%, about 20%, about 25%, about 30%, preferably at least about 40%, about 50%, more preferably at least about 60%, about 70%, or about 90%, about 100%, about 150%, or about 200%, or greater.

Alternatively, the diagnostic method can be carried out using antibodies to detect the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide or the functional fragments thereof. In one embodiment, the method includes comparing level of a suitable FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide molecule in a biological sample from a subject with a control level of the polypeptide molecule, wherein a significant change in the level of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide is an indication of the CAD in the subject. The term "significant change" refers to a change in the amount of the polypeptide or the functional fragments thereof relative to that from a biological sample of normal healthy origin, by at least about 10%, about 20%, about 25%, about 30%, preferably at least about 40%, about 50%, more preferably at least about 60%, about 70%, or about 90%, about 100%, about 150%, or about 200%, or greater.

Prognosis, stage and monitoring of coronary artery disease

In one aspect, the present invention provides methods for determining onset, prognosis and stage of CAD based on examining the expression levels of a Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid, polypeptide and/or the functional fragments thereof. As used herein, prognosis refers to the prediction of the probable course and outcome of a disease.

In general, the methods used for prognosis or stage of CAD involve comparison of the amount of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) in a sample of interest with that of a control sample to detect relative differences in the expression levels of the Farp2 or Stk25 (or a suitable homolog or ortholog thereof). The difference can be measured qualitatively and/or quantitatively. The control sample can be CAD-free or normal sample, or the sample known not to progress, or the sample known to progress. Also as used herein, the CAD stage refers to the sequence of the events, in which the CAD develops and causes symptoms. In addition, staging is a process used to describe how advanced the CAD state is in a patient. Methods of the present invention are useful in assaying the staging of CAD. The staging can be accomplished by determining the expression levels of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) relative to a reference level. The reference level can be that from CAD-free, healthy samples, or CAD samples at different stages in disease development.

The present invention further provides methods of monitoring CAD progression or recurrence in a subject by measuring over time the expression levels of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid or polypeptide or the functional fragments thereof.

In one embodiment, the methods include a) determining at a first time point the expression level of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid molecule in the subject; b) determining at a subsequent time point the expression of the Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid in the subject; and c) comparing the expression level at the first time point with that at the subsequent time point, wherein a significant change in the expression level is an indication of the onset, progression, or regression of the CAD. In another embodiment, the methods include a) determining at a first time point the expression level of a FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide molecule in the subject; b) determining at a subsequent time point the expression of the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide in the subject; and c) comparing the expression level at the first time point with that at the subsequent time point, wherein a significant change in the expression level is an indication of the onset, progression, or regression of the CAD. Increased expression levels of the Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid or polypeptide or the functional fragments thereof at the subsequent time point relative to the earlier time point, indicate that the disease is progressing to a more severe stage. In contrast, reduced expression levels at the subsequent time point indicate that the disease is progressing to a less severe stage.

Efficacy of therapy and therapeutic compound

In another aspect, the present invention also provides methods that permit assessment and/or monitoring of a patient who will be likely to benefit from both traditional and non-traditional treatments and therapies for CADs. An advantage of the present invention is the ability to monitor, screen over time, those patients who can benefit from one, or several, of the available therapies, over time to determine how the patient is faring from the treatment(s), whether a change, alteration, or cessation of treatment is warranted, or whether the patient's disease sate or stage has progressed. The identification of a correct patient for a particular therapy according to this invention can provide an increased efficacy of the treatment and can avoid subjecting the patient to unwanted and life-threatening side effects of the therapy. The ability to monitor a patient undergoing a course of therapy using the methods of the present invention can determine whether the patient is adequately responding to the therapy over time, to determine whether dosage or amount or mode of delivery should be altered or adjusted, and to ascertain whether the patient is improving during therapy, or is regressing or is entering a more severe or advanced stage of disease.

A method of monitoring according to this invention reflects the serial, or sequential, testing or analysis of a patient afflicted with a CAD or atherosclerotic condition by testing or analyzing the patient's body fluid sample over a period of time, such as during the course of treatment or therapy, or during the course of the patient's disease. For instance, in serial testing, the same patient provides a body fluid sample, e.g. , serum or plasma, or has sample taken, for the purpose of observing, checking, or examining the expression levels of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid or polypeptide or the functional fragments thereof in the patient by measuring the FARP2 or STK25 (or a suitable homolog or ortholog thereof) levels during the course of treatment, and/or during the course of the disease, according to the methods of the invention.

Similarly, a patient can be screened over time to assess the FARP2 or STK25 (or a suitable homolog or ortholog thereof) levels in a biological sample for the purposes of determining the status of his or her disease and/or the efficacy, reaction, and response to the treatment or therapy that he or she is undergoing. It will be desirable that one or more biological samples are optimally taken from a patient prior to a course of treatment or therapy, or at the start of the treatment or therapy, to assist in the analysis and evaluation of patient progress and/or response at one or more later points in time during the period that the patient is receiving treatment and undergoing clinical and medical evaluation.

Levels can be monitored over a period of days, weeks, months, years, or various intervals thereof. The patient's body fluid sample, e.g., a serum or plasma sample, is collected at intervals, as determined by the practitioner, such as a physician or clinician, to determine the FARP2 or STK25 (or a suitable homolog or ortholog thereof), lipoprotein or HDL levels in the patient compared to the levels in normal individuals over the course of treatment or disease. For example, patient samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals according to the invention. Quarterly, or more frequent monitoring of patient samples, is advisable. The levels found in the patient are compared with the levels in normal individuals, and with the patient's own levels obtained from prior testing periods, to determine treatment or disease progress or outcome.

A reduction in suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) levels over time, indicating a decrease in total plasma lipoprotein and an increase in plasma HDL-C, preferably to or near the levels found in normal individuals or lower is indicative of treatment progress or efficacy, and/or disease improvement, remission, and the like

Kits

The present invention also provides for kits that contain the necessary reagents for detection of the expression levels (either nucleic acid or polypeptide level) of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) in a biological sample. Reagents can include specific probes/primers and antibodies as described supra. Kits can also contain a control/reference value or a set of control/reference values indicating normal and various clinical progression stages of disease. In a preferred embodiment, the control/reference value or a set of control/reference values are indicative of normal and various clinical progression stages of a CAD. Moreover, kits can contain a positive control, and/or a negative control for comparison with the test sample. The negative control can contain a sample that does not have a Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid or polypeptide. The positive control can contain a sample that has various known levels of a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid or polypeptide. Kits can also contain instructions for conducting the assays and for interpreting the results. For antibody-based kits, the kits can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide or functional fragments thereof; and, optionally, (2) a second, different antibody that binds to either the FARP2 or STK25 (or a suitable homolog or ortholog thereof) polypeptide, epitope thereof, or the first antibody and is conjugated to a detectable label. For oligonucleotide-based kits, the kits can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide that hybridizes to a suitable Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid sequence or (2) a pair of primers useful for amplifying the Farp2 or Stk25 (or a suitable homolog or ortholog thereof) nucleic acid molecule. The kits can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kits can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kits can also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kits can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kits.

Such kits can be used to determine whether a subject is suffering from or at an increased risk of developing a CAD or atherosclerotic condition. Furthermore, such kits can be used to determine the prognosis, stage, or monitoring the progression of a CAD or atherosclerotic condition. Furthermore, such kits can be used for drug screening or for selection of treatment for a CAD or atherosclerotic condition.

Pharmaceutical compositions

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of a HDL modulating agent (e.g., monoclonal antibodies, or antigen-binding portion(s), antisense, siRNA, low molecular weight molecules), of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) binding molecules. For example, a pharmaceutical composition of the invention can comprise a combination of antibodies or agents that bind to different epitopes on the target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include HDL modulating agent combined with at least one other cholesterol-reducing agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the agents of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al., 1977 J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfϊte, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. 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. In many cases, one can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, from about 0.1 per cent to about 70 per cent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains 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 sensitivity in individuals. For administration of the HDL modulating agent, e.g., an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Dosage regimens for an antibody include 1 mg/kg body weight or 3 mg/kg body weight by intravenous administration, with the antibody being given using one of the following dosing schedules: every four weeks for six dosages, then every three months; every three weeks; 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more binding molecules (e.g., monoclonal antibodies) with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. The HDL modulating agent is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of HDL modulating agent (e.g., antibody) in the patient. In some methods, dosage is adjusted to achieve a plasma concentration of the antibody of about 1-1000 μg/ml and in some methods about 25-300 μg/ml. Alternatively, HDL modulating agent can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the HDL modulating agent in the patient. For example, with antibodies, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of HDL modulating agent results in a decrease in severity of disease symptoms (e.g., a decrease in plasma cholesterol, or a decrease in a symptom of a cholesterol-related disorder), an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

A composition can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for HDL modulating agent include, but are not limited to intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Alternatively, a HDL modulating agent can be administered by a nonparenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices known in the art. For example, in one embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices shown in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Examples of well known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which shows an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which shows a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which shows a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which shows a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which shows an osmotic drug delivery system having multi- chamber compartments; and U.S. Pat. No. 4,475,196, which shows an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, HDL modulating agent can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade, 1989 J. Cline Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. 5,416,016 to Low et al.); mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al., 1995 FEBS Lett. 357:140; M. Owais et al., 1995 Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., 1995 Am. J. Physiol.1233: 134); pl20 (Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen; MX. Laukkanen, 1994 FEBSLett. 346:123; JJ. Killion; I.J. Fidler, 1994 Immunomethods 4:273

EXEMPLIFICATION Example 1.

There is strong evidence that raising the plasma levels of functional HDL decreases the risk of coronary artery disease (Gotto, A. et al., J. Am. Coll. Cardiol., 43:717-724, 2004). Apoa2 was previously identified as the Chr 1 QTL gene that accounted for many, but not all of the crosses (Wang, X. et al., Genome Res., 14, 1767-1772, 2004). Soatl has been identified as the most likely QTL gene for four additional crosses; Cast x 129, B6 x KK, Pera x DBA/2, and RIII x 129. However, the following information indicated there might be a third QTL on Chr 1. First, the broad confidence interval for the C57BL/6 (B6) and 129Sl/SvImJ (129) cross indicated the presence of two closely linked QTLs, Hdlql4 and HdIq 15 (Ishimori, N. et al., Arterioscler. Thromb. Vase. Biol, 24:161-166, 2004). Apoa2 is the QTL gene for Hdlql5, but Soatl, although located within the Hdlql4 region, is unlikely to be the causal gene for Hdlql4 because Soatl does not show any expression or sequence differences between the B6 and 129 strains, indicating that a third QTL and a third QTL gene might exist. Second, the human HDL QTL at Chr 2q36.1-37.1 (FIG. 1) is homologous to this region, but does not contain either Apoa2 or Soatl, which also suggests that a third QTL might be exist in the mouse in the Hdlql4 region homologous to the human 2q QTL. To confirm HdIq 14 on chromosome (Chr) 1 identified in the cross B6 x 129 and identify the QTL gene, quantitative trait loci (QTL) analysis was performed in F2 population generated from strains NZB and NZW, which are same at Apoa2 on Chr 1 , to avoid its strong effect on the nearby QTLs. Hdlql4 was proved and the critical interval was reduced from 45 Mb harboring 225 genes to a region containing only two genes: Farp2 and Stk25. It was found in 43 genetically diverse mouse strains that strains with the FARP2 Leu821 variant had significantly higher plasma HDL levels than those with the FARP2 Pro821 variant.

The QTL for HDL on Chr 1 is complex. Of the 23 published crosses using HDL as a phenotype, 15 of them detected a QTL on Chr 1 (Rollins, J. et al, Trends Cardiovasc. Med., 16:220-234, 2006). The LOD score plot for many of these Chr 1 QTLs was broad and complex, suggesting multiple QTLs. In one cross, B6 ^χ 129, statistical evidence was obtained for at least two QTL, named Hdlql4 and Hdlql5 (Ishimori, N. et al, Arterioscler. Thromb. Vase. Biol, 24:161-166, 2004). Hdlql5, which has a peak at 104 cM, is determined by an Ala61-to-Val61 amino acid change in the Apoa2 gene; sequencing showed that inbred strains had 5 different Apoa2 genes with 9 amino acid changes among them but the only crosses that detected the QTL differed between alanine or valine at position 61. Because the Apoa2 QTL has a strong effect, it may overwhelm the impact of nearby QTLs. By examining crosses with a QTL on Chr 1 that were not explained by the Apoa2 polymorphism, it was shown that Soatl was the QTL gene in four crosses. However Soatl could not be the QTL gene for HdIq 14, detected in the B6 x 129 cross and located at cM 80, because these strains did not differ in coding region sequence or expression levels of Soatl.

Using a dense SNP map from the Broad Institute (see their website at broad.mit.edu/snp/mouse/), strains were chosen to intercross with the following characteristics: (1) no QTL at Soatl because the strains had an identical haplotype in the Soatl gene, (2) no QTL at Apoa2 because the strains had the same amino acid at position 61 in the Apoa2 gene, and (3) different haplotypes in the HdIq 14 region. Only one pair of readily available strains fit these criteria. NZW/LacJ (NZW) and NZB/BINJ (NZB) had identical haplotypes at the Soatl gene (data not shown) and both have valine at position 61 in Apoa2. NZW had a haplotype that was identical to 129 and NZB had a haplotype identical to B6 over most but not all of the 82- 162 Mb region defined by the HdIq 14 confidence intervals. For part of the 82- 162 Mb region, the haplotype of NZW is identical to NZB; if the QTL gene that causes HdIq 14 is located in these regions that appear to be identical by descent between NZW and NZB, we will not detect a QTL; if however, the QTL gene is located in the regions where NZB and NZW differ, we should be able to map this QTL without the interference of differences caused by the Apoa2 and Soatl gene.

Hdlql4 is confirmed in an intercross between strains NZB and NZW. Plasma HDL concentrations were measured in 264 (NZB x NZW) F2 male and female mice after animals were fed the high- fat diet for 8 weeks. The distribution of log transformed HDL in the F2 progeny was approximately normal (FIG. 2A). Interval mapping for Chr 1 revealed a locus influencing plasma HDL with a peak near D1MU336 (58.7 cM, 98.44 Mb in NCBI Build 36) and a significant LOD score of 3.02 (FIG. 2B, Line I). The 95% confidence interval, defined by a 1-unit decrease in LOD score on either side of the peak marker, is from 80-125 Mb, which overlapped with the 82-162 Mb HdIqH region identified in the 129 x B6 intercross. The allele for high HDL was recessive, because F2 mice that were NZW homozygous at D1MU336 had significantly higher HDL levels (Fig. 2C) compared to mice with either heterozygous genotype or homozygous NZB genotype at this locus.

HdIq 14 region is narrowed to 19 genes using bioinformatics tools. The confidence intervals for the QTL identified from these intercrosses are quite large; 80 Mb containing 487 genes for the B6 x 129 cross and 45 Mb containing 225 genes from the NZB x NZW cross (FIG. 3A and 3B, respectively). To narrow the QTL region and reduce the number of candidate genes, we used a variety of bioinformatics and statistical tools. These tools, which include combining crosses, comparative genomics, and haplotype analysis, allow a stepwise narrowing of a QTL interval, prioritizing candidate genes for further analysis. Hdlql4 is narrowed to 27.3 Mb containing 125 genes by combining crosses. The large confidence interval of a typical QTL results from the limited number of recombination events. By combining crosses, the number of recombinations is increased and the QTL interval reduced (Li, R. et al, Genetics, 169:1699-1709, 2005). The raw data for HdIqU from both the present (NZB x NZW) F2 intercross and the previous (B6 x 129) F2 intercross were combined by recoding the B6 and NZB genotypes as a single low HDL allele and 129 and NZW genotypes as a single high HDL allele. Reanalysis of the combined chromosome 1 data increased the LOD score for Hdlql4 to 5.01 (Fig. 2B, Line II), narrowed the 95% confidence interval to 27.3 Mb spanning Mb 89.8-117.1 Mb (Fig. 3C and 2B, Line II) and reduced the number of genes to 125.

Hdlql4 is narrowed to 6.2 Mb containing 89 genes by comparative genomics. Rodent and human QTLs for the same trait map to homologous genomic locations. Because the mammalian genome has been broken up and rearranged during evolution, there are about 340 homologous segments between mouse and human (LA., P., Mamm. Genome, 14:429-436, 2003). Therefore, each mouse QTL of 20 cM or so may be homologous to three or four different human chromosomes. Comparison of these homology maps may reduce the QTL size in both species if one assumes that homologous QTLs are caused by the same gene in both species; this assumption has proven true for a number of traits that were first identified in the mouse and then confirmed in humans (Stoll, M. et al, Genome Res., 10:473-482, 2000; Sugiyama, F. et al, Genomics, 71:70-77, 2001; Dwyer, J. et al., New Eng. J. Med., 350:29-37, 2004; Krude, H. et al, Nature Gen., 19:155-157, 1998; Hillebrandt, S. et al, Nature Gen., 37:835-843, 2005; Ueda, H. et al, Nature, 423:506-511, 2003; Wang, X. et al, Nature Gen., 37:365-372, 2005; Korstanje, R. & DiPetrillo, K., Am. J. Physiol Renal Physiol, 287:F347-352, 2004; Klein, R., J. Musculoskel Neuronal Int., 2:232-236, 2002). Using the genome homology between mouse and human, the HDL QTL identified on human Chr 2q37.2-37.3 was compared with Hdlql4; this human QTL is homologous only to the 84-96 Mb portion of Hdlql4. Since the combined-cross already eliminated any region proximal to 89.8 Mb, using comparative genomics narrows the distal end of the QTL, eliminating the region from 96 to 117 Mb. This step reduces the QTL region to 6.2 Mb and 89 genes (FIG. 3D).

HdIq 14 is narrowed to 2.3 Mb containing 19 genes by haplotype analysis. The haplotypes from all four parental strains were compared throughout the reduced QTL interval (Mb 89.8-96) to identify genomic regions shared among strains NZW and 129 but different from NZB and B6 strains. A common haplotype block was defined to be three or more consecutive shared alleles as illustrated in FIG. 4 (which shows only a partial analysis of the region). In defining the region that could contain a gene, the SNP above and below the endpoint was used (for example 90.35 Mb to 90.51Mb in Figure 4). The SNP map from the Broad Institute, which had 439 SNPs at an average density of one SNP/14.3 kb in the 89.8-96 Mb region, was first used and confirmed with the Perlegen database, which has many more SNPs but does not include one of the four parental strains, NZB. Haplotype analysis narrowed the QTL region to 2.3 Mb, which contained only 19 genes (FIG. 3E, Table 1). Evaluating the reduced list of candidates for expression differences. To evaluate whether any of the genes showed an expression difference between the parental strains, we used Affymetrix expression microarrays to compare the transcript profiles. These data came from another experiment (manuscript in preparation) that compared liver expression in males and females of 12 strains fed either chow or high fat diet. Three of the four parental strains of these crosses were included in the 12 strain survey; 129, B6 and NZB strains. Of the 15 candidate genes tested, all except the two Riken clones had a probe on this Affymetrix chip. Only the probes that blast correctly to one unique location were examined. Two of these 13 genes, Farp2 and Stk25, showed a significant expression difference between strains B6 and 129 (the parents of the cross). It is not possible to compare NZW and NZB because the microarray experiment does not include NZW; however, the haplotype data shows that NZW should have the same regulatory information as 129, so NZB was compared with 129. The expression difference remained for Stk25 but not for Farp2. Neither gene shows any difference in expression between B6 and NZB as expected since both have the same haplotype for HdIq 14 (Table 2). Evaluating the candidates for sequence differences. A QTL could result from a difference between parental strains in expression or in the function of protein. A functional difference would result from a sequence difference in the coding region. To evaluate whether any of these genes had a polymorphism in the coding region, we examined the SNPs in the Mouse Phenome Database on a gene by gene basis for any coding difference among any strains; these are listed in Table 3. For the eight genes with a coding region variant; the sequence changes were classified by whether the base change resulted in the same amino acid (Cs for coding region-synonymous) or in a changed amino acid (Cn for coding-nonsynonymous). Although it is expected that an amino acid needs to change to produce a functional change in the protein, evidence has shown that a synonymous change can affect the protein level and function if the frequency of the codon is changed. The slower rate of protein synthesis caused by a rare codon not only changes the level of protein but also may change the function by allowing abnormal folding and secondary structure to occur. Using the codon frequency tables (available at the website kazusa.or.jp/codon), each of the synonymous changes were evaluated; only two caused a change in the frequency of the codon. In the E030010N08Rik gene, the frequency of the codon used changed from 14% (GTT) to 23% (GTC). In the Stk25 gene the frequency of the codon used changed from 41% (ACA) to 8% (ACG) (Table 3). Each of the non-synonymous amino acid changes in the six genes were also evaluated (Table 3). No genes had a change that affected polarity, and only Glrpl had a change that affected the charge of the protein and. A change in cysteine, as in the Mterfd2 gene could change important disulfide bonds and a change in proline, as in the Farp2 gene, could affect the folding of a protein and thus its function. SNP databases, even that from the Perlegen resequencing, do not contain all the SNPs. Thus, five of these genes were sequenced to ensure that all the potential polymorphisms had been found; Hdlbp because its name, HDL binding protein, suggested that it might have a role as an HDL gene, and Snedl, Mterfd2, P ask, and Farp2 because each of them had a non-synonymous amino acid change. Each exon plus at least 50 nucleotides of the adjacent introns was sequenced for each gene. Six genes were identified to have had polymorphisms that changed an amino acid (Table 3).

The question was also addressed as to whether the changed amino acid in any of these genes was in a conserved region or known functional domain. The amino acid changes in SNEDl, MTERFD2 and PASK did not occur in conserved regions or in known functional domains. The gene Glrpl was found only in the mouse and had no homologies. The variant Pro821Leu of FARP2 (FIG. 5A) was found in region conserved among mammalian species (FIG. 5B), this region is recognized as pleckstrin homology (PH) domain (Lemmon, M., Biochemical Society symposium, 81-93, 2007), which has been identified in proteins with diverse enzymatic or regulatory functions such as phospholipases, GTPase-regulating proteins and protein kinases, as well as lipid-binding proteins (Blomberg, N. et al, Trends Biochem ScL, 24:441-445, 2007). The pleckstrin domain itself is thought to bind lipids (Klopfenstein, D. & Vale, R., MoI. Biol. Cell, 15:3729-3739, 2004). Because Farp2 was a promising candidate, the gene was sequenced in 12 strains (129, A/J, B6, C3H, CAST, DBA2, FVB, NOD, NZB, NZW, RIII, SM, and SJL) and the functional SNP (rs 13475988) was determined in an additional four strains (Pera, 1/LnJ, NZO, and NON).

Evidence from previous crosses points to Farp2 and Stk25 as the QTL gene. The evaluation of sequence and expression reduced the number of candidate genes to two high probability candidates (Stk25 with a expression difference and a large change in the codon usage (from ACA to ACG) and Farp2 with an amino acid change (Leu821Pro) in a conserved functional domain) and a few other genes that can not be completely ruled out (two Riken clones not tested for expression, Snedl, Mterfd2 and Pask with an amino acid change not in a conserved region, and Glrpl, which has an amino acid change but is a protein not shared with other mammalian species).

The cross NZO x NON was examined. These parental strains have different haplotypes in the region containing the Riken clones and the Snedl, Mterfd2, Pask, and Glrpl genes, but the same haplotype for Farp2 and Stk25 (FIG. 6B). Because this cross does not have any QTL in the Hdlql4 region (FIG. 6A), this cross does not differ in the QTL gene. This eliminates the genes in the upper part of the region and reduces the list of candidate genes to just those in the region with identical haplotypes; Farp2 and Stk25. To confirm these findings, the cross B6 x C3H was examined. This cross has the same haplotype at the Farp2 and Stk25 region (FIG. 6B); if one of these two genes is the QTL gene, there should be no QTL at cM 55.3. The LOD score plot (FIG. 6A) from this cross shows no QTL at cM 58, although the difference in Soatl between B6 and C3H and the difference in Apoa2 for both crosses result in QTLs at cM 81.6 and 92.6. Both of these lines of evidence are reasoning from the absence of a QTL, and it is always harder to be sure about negative data. The examination of the final cross provided some positive evidence, was able to further confirm this finding and probably shows the actual functional polymorphism. The Chr 1 plot from cross x DBA/2 was compared. The PERA x DBA/2 cross has additional QTL at cM 55 on Chr 1 (Fig. 6A), a comparison of the haplotypes of these two strains show that they differ at the SNPs that are in the functional domain of Farp2 and Stk, this indicates that the QTL gene must be either Farp2 or Stk25 (or both).

Mice with FARP2 821 Leu allele had higher plasma HDL concentrations than did those with FARP2 821 Pro allele. To determine whether the FARP2 821 Leu-Pro variant were associated with variant plasma HDL levels, the plasma HDL concentrations of the 43 inbred mouse strains from the Mouse Phenome Database was analyzed. To avoid the strong effect of APOA2 on HDL, the 43 strains were divided into two groups based on the key amino acid change Ala61-to-Val61 (7). In APO A2 Ala61 group (30 strains), HDL levels of strains having allele 821Leu (12 strains) were significantly higher than those of strains having allele 821 Pro (18 strains) in both male and female mice (83.1 ± 4.3 vs. 63.6 ± 5.7 mg/dL, P = 0.01, and 63.4 ± 3.3 vs. 50.5 ± 3.2 mg/dL, P = 0.009, respectively) (FIG. 7A). Similar trends were found in strains having Apoa2 Vla61 (13 strains) (113.1 ± 9.8 vs. 107.6 ± 9.8 mg/dL in males, and 98.4 ± 11. 4 vs. 79.5 ± 7.9 mg/dL in females) (FIG. 7), although their difference did not reach the statistically significant because of the small number of strains. Example 2. Materials and Methods

Animals and diet: NZW and NZB mice were obtained from The Jackson Laboratory (Bar Harbor , ME) and mated to produce the (NZB x NZW) Fl progeny, which were intercrossed to produce 272 F2 progeny. Mice were maintained in a temperature- and humidity-controlled environment with a 14h light/ 1Oh dark cycle and given unrestricted access to food and acidified water. Weanling mice were fed standard chow (18% protein rodent diet, 6% fat, product 2018; Harlan Teklad, Madison, WI) until they were eight- weeks old, and then they were fed high-fat diet containing 15% dairy fat, 1% cholesterol, and 0.5% cholic acid. Experiments were reviewed and approved by the Institutional Animal Care and Use Committee of The Jackson Laboratory.

Quantization of plasma HDL: Eight weeks after high fat diet began, animals were fasted for 4 h in the morning, blood collected from the retro-orbital sinus in tubes containing EDTA and centrifuged at 9000 rpm for 5 min. Plasma was placed into a fresh tube and frozen at -20° until analyzed. Plasma samples were thawed, vortexed, and analyzed within a week of being collected. Plasma HDL concentrations from each blood sample were measured directly, using an enzymatic reagent kit (no. 650207, Beckman Coulter) according to manufacturer's recommendations on the Synchron CX Delta System (Beckman Coulter). Genotyping: DNA were prepared from tail samples using phenol -chloroform extraction subsequent to proteinase K digestion and were resuspended in 10 mM Tris.Hcl (PH:8.0). MIT microsatellite markers DlMitl, D1MU373, D1MU212, DIMM 77, DlMitl 32, D1MU336, D1MU218, D1MU103, D1MU14, and D1MU148 that discriminate between NZB and NZW alleles were genotyped in F2 progeny using agarose gel electrophoresis (NuSieve 3: 1, FMC BioProducts, Rockland, ME). SNPs rs3717961, rs3708797, and rs3022854 were genotyped by the Allele-Typing Service at The Jackson Laboratory in conjunction with KBiosciences (Hoddesdon, UK). Reported genetic map positions are retrieved from the Mouse Genome Database (see the website at informatics.jax.org). QTL analysis: HDL values were natural log transformed: The log transform reduced the right skew in the HDL distribution but, of greater importance, it also resulted in constant variance among the crosses. When cross populations are combined for analysis purposes, if one population has a greater phenotypic variance, it will tend to dominate the results. Single loci associated with the HDL were detected by interval mapping. A multiple imputation algorithm was used to account for missing marker genotypes. One-way ANOVA was used for determining the allele effects at the peak QTL marker identified in the chromosome-wide scan for HDL levels. QTL is deemed significant if they either meet or exceed the 95% genome- wide adjusted threshold, which is assessed by permutation analysis. Analyses were carried out using Pseudomarker 1.1 software (see the website at jax.org/staff/churchill/labsite).

Statistically combining crosses: Finding repetitive QTLs in cross with different strains suggests that they may have arisen from shared ancestral alleles. The raw data on Chr 1 from the cross between B6 and 129 was combined with that from the cross using NZB and NZW mice by recoding the B6 and NZB genotypes as a single low HDL allele and 129 and NZW genotypes as a single high HDL allele (Li, R. et al, Genetics, 169:1699-1709, 2005). A LOD score was computed at 2-cM intervals across the QTL interval for each cross separately and then for both crosses combined. The combined data were analyzed with the "HDL phenotype" as standardized and "cross" as an additive covariate. Identification of Hdlql4 haplotype: SNPs for use in the haplotype analysis were obtained from the extensive public databases of SNPs such as Broad SNPs (available at their website, broad.mit.edu/snp/mouse) and Perlegen SNPs (available at their website, mouse.perlegen.com/mouse). The haplotypes from all four parental strains (NZB, NZW, B6, and 129) were compared throughout the reduced interval from Mb 89.8 to 96 to identify genomic regions shared among NZW and 129 strains contributing the allele for high HDL but different from strains NZB and B6 contributing the allele for low HDL. The data sets contained 439 SNPs spanning 89.8 - 96 Mb, and the average spacing of the SNPs was of ~14.3 kb per SNP.

Narrowing Hdlql4 using comparative genomics: A bioinformatics tool that has all the mouse genes and human genes lined up in an excel sheet was found, and the mouse and human QTL were placed in their corresponding locations. Using this tool and based on the literature, the HdIq 14 region was narrowed.

Microarray expression study: This reduced list of candidate genes are used to investigate whether there are expression differences between the strains that caused the HdIq 14 QTL using the microarray database. A set of microarray data from the livers of 12 strains that were the parents of most of the crosses that gave rise to HDL QTL was used. These strains include B6, 129 and NZB. The microarray database has samples from 3 males and 3 females of each of the 12 strains fed chow or high fat diet. The search criteria for this step would be that the gene has a significant expression difference between strains B6 and 129 (the parents of the cross) but no difference in expression between B6 and NZB (which both have the same allele for HdIqH).

Looking up HdIq 14 positional candidates if there are important sequence differences: The list of candidate genes expressed in relevant tissues are used to investigate whether there are any important sequence differences by searching SNP databases (aretha.jax.org/pub-cgi/phenome/mpdcgi?Rtn= snps/door). The coding region, the UTR, and the splice sites were examined. By now, 15 common inbred strains have been sequenced by Perlegen, and the SNPs are available on the Perlegen site and are also incorporated into other easily used sites (MGI and Ensembl). This allows us to search for sequence difference in these strains at the region of interest. Three of the 4 strains used in the crosses to determine Hdlql4 have been sequenced: B6, 129, and NZW.

Gene sequencing: Sequence differences found in SNP database were evaluated by sequencing the coding region in all the four strains NZW, 129, NZB, and B6. The genomic sequence of each gene from the B6 strain was obtained from the UCSC (genome.ucsc.edu/) mouse genome assembly, and primers were designed to amplify each exon plus at least 50 nucleotides of the adjacent introns. Purified PCR products were subjected to thermocycle sequencing, and the resulting fragments were analyzed on capillary-based machines by the Jackson Laboratory DNA Sequence Laboratory. Sequence analysis was done by aligning the sequence to the genomic B6 sequence (Sequencher version 4.1.4, GeneCodes Technology). QTL analysis on Chr 1 from crosses NZO x NON, B6 x C3H, and Pera x DBA: The genotypes and HDL data for these crosses came from other experiments in our laboratory. Analyses were carried out using Pseudomarker 1.1 software (jax.org/staff/churchill/labsite). Retrieving Data: We retrieved plasma HDL concentrations of the 43 inbred mouse strains from Mouse Phenome Database. Mice had been fed LabDiet 5K52 (6% fat) and were fasted for four hours before their blood was sampled. Plasma HDL concentrations were measured with Beckman Coulter Synchron CX5 chemistry analyzer. Analyzing Statistical Difference: Data are analyzed using Graphpad Prism

(Windows v4.00, GraphPad Software, San Diego, CA). Student's t-test was used to compare the plasma HDL concentrations among the different groups.

Table 1. Positional candidates for HdIq 14

Start Position (bp) Gene Symbol

90334314 Glrpl

90763528 ENSMUST00000057209

90901586 Sh3b_P4

94965651 Agxt

94981762 2310007B03Rιk

95011514 E030010N08Rιk

95066395 Snedl

95131866 Mterfd2

95139851 Pask

95173260 Ppplr7

95204302 Tmemlόg

95236346 Hdlbp

95309450 Sept2

95358974 Farp2

95452314 Stk25

Table 2. Farp2 Expression differences among strains B6, NZB, and 129 fed a high fat diet

Female Male

129 vs B6 129 vs NZB B6 vs NZB 129 vs B6 129 vs NZB B6 vs NZB

Affy Probe FD

ID FC FDR FC FDR FC FDR FC FDR FC FDR FC R

1424583 116 ns 116 ns 100 ns 129 ns 132 000 100 ns

1435985 100 ns 113 ns -113 ns 122 ns -108 ns -113 ns

1440799 138 000 120 ns -115 ns 156 002 127 ns -115 ns

1416770 -245 000 -215 000 114 ns -240 000 -263 000 -114 ns

FC, fold change; FDR, false discovery rates.

Table 3. Changes in codon usage and the properties of amino acids identified in HdIq 14 positional candidates

Gene Codon Codon Conserved Change in property of a a a a change

Symbol change usage region Acid-Base (129-B6)

(129-B6) change property Polarity

GAA-AAA Cn Glul7Lys No Acidic-Basic Pol-Pol

Glrpl CAG-CAC Cn GIn₅IHiS No Neu-Basic Pol-Pol

CAC-CAG Cn His79Gln No Basic -Neu Pol-Pol

E0300I0

NOSRik GTT-GTC Cs Val792Val Yes

AGG-AAG Cn Argl377Lys No Basic -Basic Pol-Pol

TGT-TGC Cs Cys288Cys No

Snedl ACT-ACC Cs Thrl090Thr No

ATC-GTC Cn UeI 123VaI No Neu-Neu Non-Non

Mterfd2 TGC-TAC Cn Cys63Tyr No Neu-Neu Pol-Pol

AAC-AGC Cn Asnl₅6Ser No Neu-Neu Pol-Pol

Pask GTG-GCG Cn Val832Ala No Neu-Neu Non-Non

CAT-CAC Cs His 1144His No

Hdlbp GAA-GAG Cs Glu710Glu No

Farp2 TTA-TTG Cs Leu785Leu Yes

CTT-CCT Cn Leu821Pro Yes Neu-Neu Non-Non

Stk25 ACA-ACG Cs Thr320Thr

Claims

CLAIMSWhat is claimed is:

1. Use of an isolated antibody or functional fragment thereof, comprising an antigen-binding region that is specific for an epitope of a polypeptide encoded by a Farp or Stk gene or a homolog or ortholog thereof, wherein the antibody or functional fragment binds to a surface receptor on a cell, and prevents or ameliorates development of a HDL-associated disease.

2. The use according to Claim 1 , wherein the Farp gene is a Farp2 gene or a homolog or ortholog thereof.

3. The use according to Claim 1, wherein the Stk gene is an Stk25 gene or a homolog or ortholog thereof

4. A method for treating a HDL-associated disease comprising administering to a subject an effective amount of the antibody or functional fragment thereof, according to Claims 1-3.

5. A pharmaceutical composition comprising an antibody or functional fragment according to any of Claims 1 -4 and a pharmaceutically acceptable carrier or excipient therefore.

6. A method for treating a HDL-associated disease comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to Claim 5.

7. Use of an isolated antibody or functional fragment thereof for the preparation of a medicament for the treatment of a HDL-associated disease, wherein the antibody or functional fragment comprising an antigen-binding region that is specific for an epitope of a polypeptide encoded by a Farp gene or a Stk gene or a homolog or ortholog thereof.

8. The use of Claim 7, wherein the Farp gene is a Farp2 gene or a homolog or ortholog thereof.

9. The use of Claim 7, wherein the Stk gene is a Stk25 gene or a homolog or ortholog thereof.

10. A transgenic animal carrying a gene encoding an antibody or functional fragment thereof according to Claim 1.

11, A method for treating a coronary artery disease or atherosclerotic condition comprising inhibiting the expression or activity of a Farp or Stk or a homolog or ortholog thereof.

12. The method of Claim 11 , wherein the Farp is a Farp2 gene or a homolog or ortholog thereof.

13. The method of Claim 11 , wherein the Stk is a Stk25 gene or a homolog or ortholog thereof.

14. The method of Claim 11 , wherein the step of inhibiting the expression or activity of a Farp or Stk further comprises inhibiting the activity using an isolated antibody or functional fragment thereof comprising an antigen-binding region that is specific for an epitope of a polypeptide encoded by a Farp or Stk gene or a homolog or ortholog thereof.

15. The method of Claim 14, wherein the isolated antibody or functional fragment thereof comprising an antigen-binding region binds to a surface receptor on a cell and prevents or ameliorates the development of a HDL-associated disease.

16. A method for treating a coronary artery disease or atherosclerotic condition comprising altering the expression or activity of Farp2 or Stk25 or a homolog or ortholog thereof.

17. A method for detecting a coronary artery disease or susceptibility to a coronary artery disease comprising detecting an allele of Farp2 or Stk25 or a homolog or ortholog thereof that is indicative of a coronary artery disease or atherosclerotic condition.

18. The method of Claim 17, wherein the allele is Leu821 or Pro821.

19. A method for determining the efficacy of treating a coronary artery disease or atherosclerotic condition comprising me treatment of a coronary artery disease or an atherosclerotic condition, and comparing the level of Farp2 or Stk25 or a homolog or ortholog thereof with a reference such that the efficacy of treating the coronary artery disease or atherosclerotic condition is determined.

20. A method of identifying an agent useful for treating a coronary artery disease or an atherosclerotic condition, wherein altering the expression or activity of FARP2 or STK25 or a homolog or ortholog thereof induces increased plasma HDL-C levels, comprising contacting a biological sample with a candidate agent and determining the level of total plasma lipoprotein or HDL-C in the sample before and after contact with the candidate agent, wherein an increase in HDL-C is indicative of an agent that is useful for treating a coronary artery disease or an atherosclerotic condition.

21. A method for identifying an agent useful for treating a coronary artery disease or atherosclerotic condition comprising contacting FARP2 or STK25 or a homolog or ortholog thereof with a candidate agent in the presence of a known substrate, wherein an altered activity of the FARP2 or STK25 or a homolog or ortholog thereof identifies the candidate agent as an agent useful for treating a coronary artery disease or an atherosclerotic condition.

22. The method of Claim 21 , wherein the contacting step is performed in a cultured cell.

23. The method of Claim 21, wherein the contacting step is performed in vivo.

24. The method of Claim 21 , wherein the Farp2 or Stk25 or a homolog or ortholog is endogenous or exogenous.

25. A method for modulating a HDL-associated disease comprising administering a modulating agent that elevates HDL-C levels in a subject.

26. The method of claim 25, wherein the HDL-associated disease is selected from the group consisting of atherosclerosis, lipid disorders, Alzheimer's disease, oxidative stress, obesity, cardiovascular disease, type II diabetes and insulin resistance.

27. The method of Claim 26, wherein the lipid disorder is selected from the group consisting of: elevated cholesterol, dyslipidemic syndrome, elevated triglycerides, dyslipidemia, dyslipoproteinemia, hyperlipidemia, familial hypercholesterolemia, and familial hypertriglyceridemia.

28. The method of Claim 25, wherein the agent alters the expression or activity of FARP2 or STK25 or a homolog or ortholog thereof.

29. The method of Claim 28, wherein the modulating agent is selected from the group consisting of a small molecule, an antisense oligonucleotide, siRNA and an antibody.

30. The method of Claim 25, wherein the HDL-associated disease is any disease in which the HDL-C levels in the subject is below the accepted normal HDL-C level.

31. The method of Claim 25, wherein the HDL-associated disease is any disease in which the HDL-C levels in the subject is below the average HDL-C level of the related population.

32. The method of Claim 25, wherein the agent is administered with a pharmaceutically acceptable carrier.