JP2011514327A - Farp2 and Stk25 and their use - Google Patents

Abstract

  Methods and compositions relating to the treatment of HDL related diseases by altering the expression or activity of FARP2 or STK25 or homologs or orthologs thereof are provided.

Description

Government Support This invention was supported in part by a grant under the American Heart Association grant AHA number: 0725905T. The United States government may have certain rights to the invention.

The present invention relates generally to methods for treating coronary artery disease or atherosclerosis. In particular, the method includes increasing high density lipoprotein levels by inhibiting and modulating Farp2 and Stk25 expression and / or activity.

BACKGROUND OF THE INVENTION Atherosclerosis is characterized by lipid accumulation, inflammatory response, cell death and fibrosis in the arterial wall. Atherosclerosis is a major factor in morbidity and mortality, especially in the United States and Western countries. Genetically controlled factors such as family history, high plasma low density lipoprotein (LDL) levels and low plasma high density lipoprotein (HDL) levels, high blood pressure, diabetes, old age, being male, and smoking, high Many risk factors are involved in the development of atherosclerosis, including lifestyle factors such as eating too much processed food with fat and lack of exercise. Because LDL cholesterol (LDL-C) is pro-atherogenic, statin drugs have been developed to reduce plasma LDL-C levels and reduce the risk of adverse cardiovascular events. Recent studies suggest that lowering LDL-C levels to levels below current guidance goals further prevents atherogenesis and reduces adverse coronary events. Statins reduced new cardiovascular adverse events by a factor of three. While this is significant, it is clear that additional treatment is required. Although there is evidence that increased HDL cholesterol (HDL-C) levels prevent atherogenesis, it does not appear that there are many effective HDL-elevating agents. Therefore, there is a need for identifying drugs that increase HDL levels.

SUMMARY OF THE INVENTION The present invention is based on the unexpected discovery that Farp2 and Stk25 and their appropriate homologs and orthologs regulate high density lipoprotein levels in plasma. The presence of Hdlq14 is confirmed by carrying out crosses between strains having the same SoatI gene and not having different key amino acids specifying Apoa2QTL but having different haplotypes in the Hdlq14 region. When this cross confirmed the presence of Hdlq14, the QTL region was narrowed by combining crosses, comparative genomics, haplotype analysis and expression and sequencing studies.

  That is, the present invention relates to a new discovery that Farp2 and Stk25, and their appropriate homologs and orthologs, regulate high density lipoprotein levels in plasma. The role of this protein that affects plasma lipoprotein levels is still unknown. The present invention increases plasma HDL cholesterol levels by using Farp2 and Stk25 to affect plasma levels of HDL cholesterol, in particular, by regulating Farp2 activity and inhibiting Stk25 activity, and atherosclerosis It can be treated.

  Accordingly, in one aspect, the invention provides an isolated antibody comprising an antigen binding region that is specific for an epitope of a polypeptide encoded by a Farp gene (eg, Farp2 gene) or Stk gene (eg, Stk25 gene) or Use of the functional fragment, wherein the antibody or functional fragment relates to a use that binds to a surface receptor on a cell and prevents or ameliorates the development of an HDL-related disease.

  In one embodiment, the invention relates to a method of treating HDL-related diseases comprising administering to a subject an effective amount of an antibody or functional fragment thereof.

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

  The present invention also relates to the use of an isolated antibody or a functional fragment thereof for the manufacture of a medicament for treating HDL-related diseases, wherein the antibody or functional fragment is a Farp gene or Stk gene, or an ortholog or homolog thereof. It relates to a use comprising an antigen-binding region specific for an epitope of a polypeptide encoded by (for example the Farp2 or Stk25 gene, or an ortholog or homolog thereof).

  In one embodiment, the present invention relates to a method of treating coronary artery disease or atherosclerosis comprising modulating Farp or Stk expression. In one particular embodiment, Farp is the Farp2 gene and Stk is the Stk25 gene. In another embodiment, the inhibition of Farp or Stk expression and / or activity further comprises a poly gene encoded by a Farp gene (eg, Farp2 gene) or Stk gene (eg, Stk25 gene), or a homolog or ortholog thereof. Inhibiting activity with an isolated antibody or functional fragment thereof comprising an antigen binding region specific for an epitope of a peptide. In particular, an isolated antibody or functional fragment thereof comprising an antigen binding region binds to a surface receptor on the cell and prevents or ameliorates the development of HDL-related diseases. Also included within the scope of the invention is a transgenic animal having a gene encoding an antibody or functional fragment.

  In one embodiment, the invention relates to a method for treating coronary artery disease or atherosclerosis comprising altering the expression or activity of Farp2 or Stk25 or a homolog or ortholog thereof.

  In another embodiment, the present invention relates to a method for detecting susceptibility to coronary artery disease or coronary artery disease, comprising detecting an allele of Farp2 or Stk25, which is an indicator of coronary artery disease or atherosclerosis, or a homolog or ortholog thereof. About. In one particular embodiment, the allele is FARP2 Leu821 or Pro821.

  In another embodiment, the present invention is a method for measuring the efficacy of treatment of coronary artery disease or atherosclerosis, treating coronary artery disease or atherosclerosis, and treating coronary artery disease or atherosclerosis. It relates to a method comprising comparing the level of Farp2 or Stk25 or a homolog or ortholog thereof to a control so that the efficacy of the treatment is determined.

  In another embodiment, the present invention is directed to the treatment of coronary artery disease or atherosclerosis that induces an increase in plasma HDL-C levels by altering the expression or activity of Farp2 or Stk25 or a homolog or ortholog thereof. A method of identifying a useful agent comprising contacting a biological sample with a candidate agent and measuring the level of total plasma lipoprotein or HDL-C in the sample before and after contact with the candidate agent, Is an index of a drug useful for the treatment of coronary artery disease or atherosclerosis.

  In another embodiment, the present invention provides a method for identifying a drug useful for the treatment of coronary artery disease or atherosclerosis, wherein FARP2 or STK25 or a homolog or ortholog thereof is contacted with a candidate drug in the presence of a known substrate. And a method for identifying candidate agents as agents useful for the treatment of coronary artery disease or atherosclerosis by altering the activity of FARP2 or STK25 or a homologue or orthologue thereof. In one particular embodiment, the contacting step is performed on cultured cells. In another embodiment, the contacting step is performed in vivo. In another embodiment, FARP2 or a homolog or ortholog thereof is endogenous or exogenous.

  In another embodiment, the present invention relates to a method of modulating HDL-related disease comprising administering a modulating agent that increases HDL-C levels in a subject. In one embodiment, the HDL-related disease is selected from the group consisting of atherosclerosis, lipid disorders, Alzheimer's disease, oxidative stress, obesity, cardiovascular disease, type 2 diabetes and insulin resistance. In another embodiment, the lipid disorder is a group consisting of hypercholesterolemia, dyslipidemia syndrome, hypertriglyceride, dyslipidemia, dyslipidemia, hyperlipidemia, familial hypercholesterolemia and familial hypertriglyceridemia Selected from. In one particular embodiment, the agent alters the expression or activity of FARP2 or STK25 or a homolog or ortholog thereof. In one particular embodiment, the modulating agent is selected from the group consisting of small molecules, antisense oligonucleotides, siRNA and antibodies. In one particular embodiment, the HDL-related disease is a disease in which the subject's HDL-C level is below a generally accepted normal HDL-C level. In one particular embodiment, the HDL-related disease is a disease in which the subject's HDL-C level is below the mean HDL-C level of the relevant population. In one particular embodiment, the agent is administered with a pharmaceutically acceptable carrier.

FIG. 1 is a Chr. 1 chromosome map of mouse HDL QTL and their human-matched QTL. The vertical line represents the chromosome, with the centromere on the top. Mouse HDL QTL is represented by the bar on the left side of the chromosome. Each bar represents QTL from one hybrid. QTL size is 95% confidence interval (CI), 1.5-LOD drop interval (if 95% CI is not valid), or ± 10 cM around LOD score peak (if neither CI nor LOD score numbers are valid) Is given as either. The mouse homology region of human HDL QTL is represented by the bar on the right side of the chromosome, and the chromosome number of these human QTLs is appended to their right side.

FIG. 2 shows that Hdlq14 is confirmed in the NZB × NZW cross. (A) Distribution of log-transformed plasma HDL concentrations of 264 male and female F2 offspring. (B) Detailed LOD scores are shown for plasma HDL at Chr1 from the hybrid NZB × NZW (black line), and Hdlq14 is reduced by combining the hybrids (blue line). The X axis shows the marker position in centimorgan units and the Y axis shows the LOD score. In the hybrid combination, data on Hdlq14 from both the current (NZB × NZW) F2 hybrid and the previous (B6 × 129) F2 hybrid were combined by narrowing QTL using statistical techniques. The second peak at cM92 in the blue line represents QTL due to the Apoa2 gene difference between B6 and 129. (C) Allele effect at peak marker D1Mit336 in plasma HDL. WW, BB and BW indicate F2 mice that are homozygous and heterozygous for the NZW allele and the NZB allele, respectively, at the marker locus. Values are expressed as the mean HDL ± SEM of F2 having a specific genotype at the indicated locus (P = 0.008 and 0.0002 in males and females, respectively).

FIG. 3 shows the Hdlq14 region narrowed by different strategies. (A) 95% CI is between Mb 82-162 in B6 × 129 hybrid. (B) 95% CI is between Mb 80-125 in NZB × NZW hybrids. (C) Hdlq14 was reduced to the 125-gene 27.3-Mb region (Mb 89.8 to 117.1) by combining the hybrids. (D) Using hybrids NZB × NZW and B6 × 129 that detected Hdlq14, the region was reduced to 2.3 Mb containing 19 genes by haplotype analysis. (E) Using hybrids B6 × C3H and NON × NZO that could not detect Hdlq14, QTL was further reduced to 2 genes by haplotype analysis.

FIG. 4 is a fine haplotype analysis map of the region ranging from Mb 89.8 to 96 in Chr1 between 129, B6, NZB and NZW strains. The vertical row of physical positions shows the distance in megabases from the centromere according to the Ensembl database (NCBI build 36). The mouse strain is shown at the top of the figure. SNPs are indicated as nucleotides A, T, G and C observed at each position between strains. A SNP written in lower case means that the prediction is based on the surrounding haplotype. Genomic regions shared between the 129 and NZW lines are highlighted in green, and regions different from those shared by the B6 and 129 lines are highlighted in yellow. A common haplotype block is defined as being 3 or more conservative shared alleles. The common SNP is shown as a boxed portion according to the co-inheritance hypothesis.

FIG. 5 shows the sequence comparison. (A) Pro821Leu responsible for the polymorphism was identified in 16 lines by sequencing. Allele C (top) is identified in the B6, C3H, CAST, PERA, NON, NZB, NZO, RIII and SM lines, and allele T (bottom) is 129, A / J, DBA2, FVB, I / Identified in LnJ, NOD, NZW and SJL strains. (B) Proline 821 in FARP2 is in a conserved region between mammalian species.

FIG. 6 shows the position and sequence data. (A) LOD scores are plotted as Chr1 in hybrids NZB × NZW (black), NZO × NON (orange), B6 × C3H (red) and Pera × DBA (blue). The hybrids NON × NZO and B6 × C3H do not have Hdlq14, and the hybrid PERA × DBA2 has QTL at that position. (B) Haplotype map for the region with genes E030010N08Rik, Sned1, Mterfd2, Pask, Farp2 and Stk25 between NZO and NON, B6 and C3H and PERA and DBA / 2 strain pairs. The haplotypes in the hybrids NON × NZO and B6 × C3H are the same for the bottom 5 SNPs, including the Cn SNP at Farp2 (955.4480 Mb) and all SNPs at Stk25. However, the SNPs above the bottom 5 are all different. The hybrid PERA × DBA2 is different in the bottom five SNPs.

FIG. 7 shows HDL concentrations in inbred mouse strains with FARP2 mutant Leu821 and Pro821 in background APOA2 Ala61 (upper panel) and Val61 (lower panel), respectively. Upper panel: 30 lines were divided into 2 types (12 lines with FARP2 Leu821 and 18 lines with FARP2 Pro821). Lower panel: 11 lines were divided into 2 types (6 lines with FARP2 Leu821 and 5 lines with FARP2 Pro821). Plasma HDL concentrations (mean ± SEM, mg / dl) were obtained from groups of 7-10 week old females (2 rows on the left) and males (2 rows on the right) fasted for 4 hours (Mouse Phenome Database). Each group consisted of 10-40 mice (24 ± 7 males and 22 ± 6 females, mean ± SD).

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the presence of Hdlq14 on chromosome 1 and the unexpected discovery that Farp2 and Stk25 are genes that appear to be fundamental for the quantitative trait locus (QTL) Hdlq14. Upon further testing, the FARP2 Leu ⁸²¹ variant had significantly higher plasma HDL levels than the FARP2 Pro ⁸²¹ variant.

  Cardiovascular disease is a leading cause of morbidity and mortality in the United States. About 8% of CVD occurs in Americans under the age of 50. Approximately 40% of early-onset CVD patients are estimated to exhibit low high density lipoprotein cholesterol (HDL), which represents the most common lipoprotein disease in CVD patients (Genest, J. et al., Circulation 85: 2025-2033, 1992). This inverse relationship between HDL levels and CVD has drawn interest in genetic factors that contribute to fluctuations in HDL levels. Because of the inherent difficulties in performing linkage analysis for HDL in humans, many mammalian geneticists have turned to mouse models. The use of mouse inbreeding lines in this setting is a viable alternative to human genetic studies, given the degree of reference that can be affected for experimental parameters such as environment, mating scheme and detailed phenotypic analysis. It was found (Allayee, H. et al., Front. Biosci., 11: 1216-1226, 2006).

  Over 100 QTLs affecting plasma HDL levels have been identified in mice, the most frequent being identified on distal chromosome 1 due to Apoa2 gene mutations in each of the hybrids. Disclosed herein is another QTL confirmation on proximal chromosome 1 with respect to plasma HDL levels by controlling for the major gene effect of Apoa2 in NZB × NZW hybrids. The peak at this locus is at D1Mit336 (58.7 cM, 98.4 Mb) and the 95% confidence interval is from 80 Mb to 125 Mb overlapping with Hdlq14 previously identified in hybrid B6 × 129. Syntenic analysis between mice and humans showed that the portion of the chromosomal region where Hdlq14 spreads in mice is homologous to the 2q37.2-37.3 chromosomal region in humans. In humans, plasma HDL is linked to chromosome 2q37.2-37.3 (Elbein, S. & Hasstedt, S., Diabetes, 51: 528-535, 2002) and is therefore the gene underlying HDLq14. Appears to affect HDL level changes in both mice and humans, and this hypothesis is due to other traits such as atherosclerosis (Wang, X. et al., Nature Gen., 37: 365-372 , 2005), body fat (Krude, 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, 71: 70-77, 2001).

  QTLs identified from hybrids often have large confidence intervals because they are detected from limited recombination events (Korstanje, R. & Paigen, B., Nature Gen., 31: 235 -236, 2002). By using several mating strategies, for example, selective gene analysis, recombinant progeny testing, interval-specific common gene systems, forward hybrid systems (Darvasi, A. & Soller, M., Genetics, 141: 1199-1207 , 1995), recombinant inbred segregation test, recombinant inbred mating test, and genetic heterologous stock (Mott, R. et al., Proc. Natl. Acad. Sci. USA, 97: 12649-12654; 2000; QTL can be subdivided (Darvasi, A., Nature Gen., 18: 19-24, 1998; McPeek, M. et al., Talbot, C. et al., Nature Gen., 21, 305-308, 1999). , Proc. Natl. Acad. Sci. USA, 97: 12389-12390, 2000; Flint, J. et al., Nature Rev., 6: 271-286, 2005). Bioinformatics tools are a quick and economical way to narrow QTL (DiPetrillo, K. et al., Trends Genet., 21: 683-692, 2005) and access to public sequences, genotypes and expression databases This was made possible by recent investments and new developments in statistical methods. These tools, including a combination of mating, comparative genomics and haplotype analysis, gradually reduce the QTL mapping interval and prioritize candidate genes for further analysis with the potential to identify the most promising candidate genes It became possible to do. By using this bioinformatics method, the chromosomal region from 45 Mb where Hdlq14 shows spread in the hybrid NZB × NZW is effectively reduced to 2.3 Mb, and the number of positional candidates is reduced from 225 to 19 that can be tested. It was. In the second stage, we used the parental haplotypes in the two hybrids that could not detect QTL, the NZO × NON and B6 × C3H hybrids. Since the manuscripts for these two hybrids are still being prepared (Su, Paigen), the unprocessed genotype and phenotype data are http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn= Stored in projects / qtlprojlist. For these two hybrids, we searched for haplotypes that were identical for all four lines because no QTL was detected. These hybrids had the same haplotype for SNPs in the Farp2 and Stk25 genes, but the haplotypes for the other 17 genes were different. That is, these two hybrids reduced the QTL region to only 2 genes and excluded the other 17 genes (FIG. 3, step e).

For Hdlq14 positional candidate genes, priorities were further determined by gene sequencing and expression analysis. QTL arises from differences between parental strains in protein quantity or protein function. Therefore, identifying sequence polymorphisms among the lines used for QTL detection is important in determining the causative gene. Although functional consequences are not always clear, functional differences in proteins arise from sequence changes in the coding region of DNA. Comparing the complete coding sequences of these 15 positional candidate genes from B6, 129, NZB and NZW, only 6 genes, Glrp1, E030010N08Rik, Sned1, Mterfd2, Pask and Farp2 have polymorphisms that change amino acids Was found. All positional candidate genes were examined by their differential expression between 129, B6 and NZB resulting in Hdlq14 QTL. Stk25 has significant expression differences between parental lines B6 and 129 or 129 and NZB lines with different alleles for Hdlq14, and no expression difference between B6 and NZB, both of which have the same allele for Hdlq14 It was found. This evaluation of expression differences indicates that Stk25 is a realistic candidate gene. Farp2 was not completely consistent across strains, had some expression differences, and had a non-synonymous coding region difference (Leu821Pro) in the conserved region. Haplotype data indicates that NZW and 129 have the same regulatory information as A / J at Stk25, but are different in the B6 and NZB strains. The eQTL test in a cross between A / J and B6 strains shows that Stk25 is regulated by that eQTL, and HDL levels show B6 homozygotes in hybrids derived from B6 and A / J strains. Mice with A / J homozygotes are significantly higher than those with mice (52.1 ± 8.9 vs 44.8 ± 7.9, p = 8.8 × 10 ⁻⁶ ) The J, 129 and NZW lines give the same high allele for HDL levels in different hybrids. That is, Stk25 expression appears to be regulated by itself between 129 and B6, NZW and NZB strains. Stk25 encodes serine / threonine kinase 25, which is a very important regulator of AMPK activation in muscle and hepatocytes (Imai, K. et al., Biochem. Biophys. Res. Commun., 351: 595-601, 2006), therefore, its activity is also important for our understanding of lipid metabolism.

  Mutants that change amino acids were identified in the genes Glrp1, E030010N08Rik, Sned1, Mterfd2 and Pask, while HDL QTL identified in the hybrids NZO × NOD, B6 × C3H and Pera × DBA are polymorphic in these genes. It did not seem to be attributed. The polymorphic Pro821Leu of FARP2 is in the conserved plextrin homology (PH) domain (Lemmon, M., Biochem. Soc. Symp., 81-93, 2007) and is itself thought to bind lipids (Klopfenstein) , D. & Vale, R., Mol. Biol Cell, 15: 3729-3739, 2004). It was found that mouse strains with the Leu821 variant have significantly higher plasma HDL levels than those with the Pro821 variant. These findings confirm the existence of Farp2 as the underlying Hdlq14 gene and suggest that the Farp2 polymorphism is involved in the Hdlq14 phenotype.

  Because Farp2 and Stk25 are very close to each other in physical location, it is difficult to separate them using traditional genetic strategies such as congenic mice, and the haplotype is identical between all inbred lines. It is also unthinkable to isolate them by finding crossings in different hybrids.

Definitions As used herein, the term “HDL” refers to high density lipoprotein. HDL contains approximately equal amounts of lipid and protein complexes that function as cholesterol transporters in the blood. HDL is synthesized and secreted primarily by the endothelial cells of the liver and small intestine. Immediately after secretion, HDL takes the form of discoidal particles containing apoprotein AI (also called apoA-I) and phospholipids as its main components and is also called nascent HDL. This nascent HDL receives free cholesterol from the plasma membrane of peripheral cells or cholesterol produced by hydrolysis of other lipoproteins in the blood, and the above-mentioned action is caused by the action of LCAT (lecithin cholesterol acyltransferase) at its hydrophobic center. Forming mature spherical HDL while retaining cholesterol ester converted from cholesterol. HDL plays a very important role in the physiological function related to lipid metabolism called the “reverse cholesterol transport system” that takes up excess cholesterol from peripheral tissues in the blood and transports it to the liver. Since the reverse cholesterol transport system is thought to induce a prophylactic effect against atherosclerosis, high levels of HDL reduce the risk of atherosclerosis and coronary heart disease (CHD).

  As used herein, the term “HDL modulating agent” refers to a molecule that can alter HDL expression or functional levels such that alteration of HDL levels alters HDL-related diseases or conditions. HDL modulating agents can act directly or indirectly with nucleic acids or polypeptides that affect 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 comprises an antigen binding region specific for an epitope of a polypeptide encoded by a Farp gene (eg, Farp2 gene) or Stk gene (eg, Stk25 gene) or a homolog or ortholog thereof. An isolated antibody or functional fragment thereof. Antibodies or functional fragments can prevent or ameliorate the development of HDL-related diseases by binding to surface receptors on cells.

  As used herein, the term “biological sample” refers to an entire organism or a tissue, cell or component thereof (eg, but not limited to blood, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, A subset of bodily fluids including saliva, amniotic fluid, umbilical cord blood, urine, vaginal fluid and semen. Furthermore, a “biological sample” is a whole organism or a subset of its tissues, cells or components, or, but not limited to, plasma, serum, spinal fluid, lymph, skin, respiratory, intestinal and urogenital organ surfaces Refers to a homogenate, lysate or extract prepared from a fraction comprising a slice, tears, saliva, milk, blood cells, tumor, organ or part thereof. In most cases, the sample is removed from the animal, but the term “biological sample” can refer to cells or tissues that are analyzed in vivo, eg, without being collected from the animal. Typically, a “biological sample” includes cells from an animal, but the term can also refer to non-cellular biological materials such as non-cellular fractions of blood, saliva or urine, which are cancerous. It can be used to measure related polynucleotide or polypeptide levels. Furthermore, “biological sample” also includes a medium, such as a nutrient broth or gel in which the organism has been grown, containing cellular components such as proteins or nucleic acid molecules.

  As used herein, the term “nucleic acid” includes deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. This term refers to known nucleotide analogs or modified backbone residues or bonds that exist synthetically, naturally and non-naturally, have binding properties similar to reference nucleic acids, and are metabolized in the same way as reference nucleotides. Includes nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methylribonucleotide and peptide-nucleic acid (PNA). Nucleic acid sequences also include naturally occurring allelic variants of the above nucleic acids.

  As used herein, the term “oligonucleotide” comprises a nucleic acid molecule consisting of two or more deoxyribonucleotides or ribonucleotides linked by phosphodiester bonds, preferably comprising about 6 to about 300 nucleotides in length. Say. The size of the oligonucleotide depends on many factors, including the ultimate function or use of the oligonucleotide. Preferably, for example, an oligonucleotide that functions as an extension primer is long enough to initiate synthesis of the extension product in the presence of a catalyst, such as DNA polymerase, and deoxynucleotide triphosphate. Furthermore, as used herein, the term “oligonucleotide” also encompasses oligonucleotides that are structurally modified (“modified oligonucleotides”) but function similarly to unmodified oligonucleotides. Modified oligonucleotides can include non-natural moieties such as modified sugar moieties or intersugar linkages, such as phosphorothioates.

  As used herein, the term “polypeptide” refers to a polymer in which the monomers are amino acids and are linked by peptides or disulfide bonds. The term also encompasses a full length natural amino acid sequence or fragment thereof of about 8 to about 500 amino acids in length. In addition, unnatural amino acids such as beta-alanine, phenyl, glycine and homoarginine may also be included. All amino acids used in the present invention can be D- or L-optical isomers. Polypeptide sequences also include naturally occurring allelic variants of the above polypeptides.

  As used herein, “quantitative trait locus” (QTL) analysis refers to a means of discovering new genes that regulate complex properties (Abiola, O. et al., Nature Rev., 4: 911). -916, 2003). QTL analysis is particularly important for biomedical research because the location of human disease QTL can often be predicted from the QTL detected in mouse disease models. This position of disease QTL in the homology region for both mice and humans suggests that the same gene regulates these properties in both species. That is, QTL analysis using a mouse model can potentially identify genes important in human disease.

  As used herein, the term “subject” includes animals. Preferably, the animal is a human or non-human mammal. Also preferably, the subject is, for example, a primate (eg, 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 extent to which a desired effect is obtained. Specifically, the term refers to the extent to which plasma lipoprotein and HDL levels are modulated (eg, increased, increased, inhibited, reduced or delayed). The term “efficacy” as used herein also refers to the reduction or reduction of one or more symptoms or clinical events of coronary artery disease (CAD). Symptom reduction or reduction includes, but is not limited to, reduction of clinical events such as phosphatidylcholine degradation, reduction or elimination of oxidized phospholipids, reduction of atherosclerotic plaque formation and failure, heart attack, angina or stroke , Decreased hypertension, decreased inflammatory mediator biosynthesis, decreased plasma cholesterol. Symptom reduction or reduction can also include improved blood flow to the vascular bed affected by atherosclerosis.

  As used herein, the term “coronary artery disease” (CAD) or synonymous “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 gradually with arterial stenosis due to intimal hyperplasia and plaque formation. As the plaque thickens, the arteries narrow and blood flow is reduced, reducing oxygen supply to the myocardium. This reduction in blood flow has a series of significant consequences for the myocardium. For example, interruption of blood flow to the myocardium induces an “infarction” (myocardial infarction), commonly known as a heart attack. Those clinical signs and symptoms resulting from the blockage of arteries delivered to the heart are the manifestations of CAD. CAD manifestations include angina, ischemia, myocardial infarction, cardiomyopathy, congestive heart failure, arrhythmia and aneurysm formation. It can be seen that fragile plaque disease in the coronary circulatory system is causally associated with distal embolization that manifests as arterial thrombosis or itself myocardial infarction. CAD can encompass a series of stages. The early stages of CAD are characterized by atheroma lines that occur in the walls of the coronary arteries that do not block blood flow. Over a period of years, these streaks thicken. That is, the next stage of CAD is characterized by the formation of plaques that expand into the arterial wall and vascular lumen and affect blood flow through the artery. A subject is said to have developed symptoms of obstructive CAD or ischemic heart disease when the plaque increases in thickness and blocks most of the vessel diameter. Symptoms often include exertional angina or decreased exercise tolerance. As the degree of CAD progresses, the lumen of the coronary artery is almost completely blocked, and the blood flow that delivers oxygen to the myocardium can be severely restricted. This stage of CAD is called myocardial infarction (heart attack) and is characterized by signs and symptoms of chronic coronary ischemia, including symptoms of resting angina and acute pulmonary edema.

  As used herein, the term “atherosclerosis” refers to one or several arteries and small arteries of the body and body organs, such as but not limited to coronary arteries, aorta, Abnormal accumulation of cholesterol and cholesterol esters and related lipids in macrophages, smooth muscle cells and other types of cells leading to stenosis and / or occlusion of arteries supplying blood to the renal arteries, carotid arteries, limbs and central nervous system This is the process. Atherosclerosis is completely insidious that lasts for decades until the atherosclerotic lesions have collapsed due to physical forces from the bloodstream and the deep arterial wall components are exposed to flowing blood. And can induce thrombosis and interfere with oxygen supply to target organs such as the heart or brain. According to one theory, atherosclerosis has 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 cells Growth, and 5) synthesis of extracellular matrix. In addition, relevant inflammatory responses and mediators of this pathological process are also included in this definition.

  As used herein, “HDL-related” disease refers to a disease or trait with low HDL levels, such as atherosclerosis. These related diseases include, for example, atherosclerosis, lipid disorders, Alzheimer's disease, oxidative stress, obesity, cardiovascular disease, type 2 diabetes and insulin resistance (also referred to as metabolic syndrome). Examples of lipid disorders include cholesterol elevation (LDL levels exceeding 130 milligrams per milliliter or mg / dL), dyslipidemia syndrome, triglyceride elevation (high triglyceride levels as high as 1500 mg / dL), dyslipidemia or abnormal lipoprotein blood. Symptoms (HDL is less than 35 mg / dL), hyperlipidemia or high cholesterol, familial hypercholesterolemia (genetic disease that increases total and LDL cholesterol) and familial hypertriglyceridemia (genetic hypertriglyceride) Can be mentioned.

  As used herein, the expression “significant change in expression level” refers to an increase or decrease in expression level from a control level in an amount greater than the standard error of the assay used to evaluate expression. This expression is also 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 more.

  As used herein, the term “gene” refers to a nucleic acid sequence that encodes a polypeptide and regulates its expression. Thus, a gene includes regulatory elements such as promoters, splice sites, enhancers, repressor binding sites, and the like. A gene can have many different “alleles” that can affect the polypeptide sequence or expression level, or are sequence variants that 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 endogenous or exogenous.

  As used herein, the term “expression level” (eg, Farp2 or Stk25) refers to the amount of mRNA transcribed from a corresponding gene present in a biological sample. The expression level can be detected with or without comparison with the level from the control sample or the predicted level of the control sample.

  As used herein, the term “control level” refers to a standard level of a biomarker for measuring changes. In one embodiment, a “control level” is a biomarker nucleic acid expression from a normal or healthy cell, tissue or subject, or from a population of normal or healthy cells, tissue or subject, or a biomarker polypeptide, or biomarker There may be a normal level of biological activity. As a non-limiting example, the control level can be a level of Farp2 or Stk25 polypeptide or biological activity in normal cells, tissues or subjects, or plasma total lipoprotein or HDL levels.

  As used herein, the term “control expression level” (eg, Farp2 or Stk25) refers to the amount of mRNA transcribed from a corresponding gene present in a biological sample representative of a healthy subject. The term “control expression level” can also mean an established mRNA level representative of a previously established healthy population based on measurements from healthy subjects.

  As used herein, “detect” refers to identifying the presence or absence of a molecule in a sample. If the molecule to be detected is a polypeptide, the detection step can be performed, for example, by coupling the polypeptide to a detectably labeled antibody. A detectable label is a molecule that can emit an observable signal independent of or in response to a stimulator. The 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” the label include, for example, standard or confocal microscopy, quantitative and qualitative methods adapted to FACS analysis, and the same methods adapted to high-throughput methods involving multiwell plates, arrays or microarrays There is. One skilled in the art would be able to select an appropriate filter set and excitation energy source for detecting fluorescence emission from a given fluorescent polypeptide or dye. As used herein, “detecting” can also include the use of multiple antibodies to the polypeptide to be detected, wherein the multiple antibodies bind to different epitopes on the polypeptide to be detected. The antibodies used in this method may utilize two or more detectable labels, for example including FRET pairs. If the level of detectable signal is actually greater than the background level of the detectable label, or if the measured polypeptide level is actually greater than the level measured in the control sample, the polypeptide molecule is "Detected" according to

  “Detecting” as used herein also measures or observes the signal emitted by, for example, a directly or indirectly labeled probe nucleic acid molecule (which can hybridize to a target in a serum sample). It refers to identifying the presence of a target nucleic acid molecule by a method. Detection of a probe nucleic acid is a direct detection of the presence of a sequence encoding a target nucleic acid, eg a marker gene, ie its detection. Methods and techniques for “detecting” fluorescent, radioactive and other chemical labels are reported by Ausubel et al. (1995, Short Protocols in Molecular Biology, 3rd edition, John Wiley and Sons, Inc.). ing.

  Alternatively, a moiety is attached to a probe nucleic acid that hybridizes to the target, which moiety can be, for example, an enzyme activity or antibody or other specific indicator that allows detection of the target in the presence of an appropriate substrate. Nucleic acids that contain specific antigens or other markers that allow detection by addition can be “indirectly detected”. Alternatively, the target nucleic acid molecule can be detected by amplifying a nucleic acid sample prepared from a patient clinical sample using oligonucleotide primers specifically designed to hybridize with a portion of the target nucleic acid sequence. . It is also possible to “detect” a target nucleic acid according to the present invention using a quantitative amplification method such as, but not limited to, TaqMan®. A nucleic acid molecule is used herein if the level of nucleic acid measured (eg, by quantitative PCR) or the level of detectable signal provided by the detectable label is actually above the background level. "Detected" as it is.

  In addition, “detect” as used herein refers to at least early detection of CAD, such as atherosclerosis, in a subject, and “early” detection preferably refers to a point before symptoms are visible. This means detecting CAD in the initial stage of the above. Further, “detect” as used herein refers to detection of CAD recurrence in a subject by using the same detection criteria as set forth above. Furthermore, “detect” as used herein refers to the measurement of the change in the extent of CAD before and after treatment with a therapeutic compound. In this case, the change in the extent of CAD in response to the therapeutic compound is one or more of the presented in the clinical sample in response to the presence of the therapeutic compound relative to the expression level in the absence of the therapeutic compound. Refers to more marker gene expression, or alternatively a percentage increase or decrease of at least about 10% of the amount of marker gene polypeptide.

  As used herein, the term “antibody” refers to an intact antibody or an antigen-binding fragment thereof (ie, “antigen-binding portion”) or a single chain (ie, light or heavy chain). An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2 and CH3. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes one domain, CL. Furthermore, the VH and VL regions can be divided into hypervariable regions called complementarity determining regions (CDRs) interspersed with highly conserved regions called framework regions (FR). Each VH and VL contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant region of the antibody can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q).

  As used herein, the term “antigen-binding portion” of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; F (ab) 2 fragments, by disulfide bridges in the hinge region A bivalent fragment containing two linked Fab fragments (generally one from the heavy chain and one from the light chain); an Fd fragment consisting of the VH and CH1 domains; consisting of the VL and VH domains of a single arm of the antibody There is an Fv fragment; a single domain antibody (dAb) fragment consisting of a VH domain (Ward et al., 1989 Nature 341: 544-546); and an isolated complementarity determining region (CDR).

  In addition, the two domains VL and VH of the Fv fragment are encoded by separate genes, which are also known as monovalent molecules (single chain Fv (scFv)) in which the VL and VH regions pair together. For example, Bird et al., 1988 Science 242: 423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85: 5879-5883). They can be linked using recombinant techniques with artificial peptide linkers. Single chain antibodies as described above comprise one or more “antigen-binding portions” of an antibody. These antibody fragments are obtained using conventional techniques well known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies.

  Antigen binding moieties can also be incorporated into single domain antibodies, maxibodies, minibodies, intracellularly expressed antibodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (eg, Hollinger and Hudson, 2005 , Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of the antibody can be grafted into a scaffold based on a polypeptide such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

  The antigen binding portion may be incorporated into a single chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) together with a complementary light chain polypeptide to form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8 (10): 1057-1062; and US Pat. No. 5,641,870).

The expressions “silence” and “inhibit expression of” are used herein to transcribe the Farp2 or Stk25 gene and to express the Farp2 or Stk25 gene as long as they are used for the target gene, eg, Farp2 or Stk25. The amount of mRNA transcribed from the Farp2 or Stk25 gene that can be isolated from the first cell or group of cells that has been treated to inhibit is substantially the same as the first cell or group of cells, The above treatment shall mean at least partial suppression of Farp2 or Stk25 gene expression as evidenced by a reduction in the amount compared to the case of a second cell or cell group (control cells) that has not been treated. The degree of inhibition is usually expressed by the following formula.

  Alternatively, the degree of inhibition is a parameter functionally linked to Farp2 or Stk25 gene transcription, such as the amount of protein encoded by the Farp2 or Stk25 gene secreted by the cell, or a cell exhibiting a certain phenotype It can be given by a reduction of the number. In principle, Farp2 or Stk25 gene silencing can be measured by appropriate assays in cells that express the target constitutively or by genomic engineering.

  The articles “a”, “an”, “the” and similar terms used herein in the English language specification of the invention (especially in the claims) are used herein Unless specifically stated otherwise or clearly inconsistent with the context, both the singular and plural cases are considered to be included. Although a range of values is listed herein, this merely serves as a short way to individually indicate each independent value contained within that range. Unless stated otherwise specifically in the specification, each individual value shall be incorporated into the specification as if it were individually recited in the specification. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of all examples or exemplary expressions (eg, “eg, etc.”) described herein are intended only to make the present invention more understandable, and may be claimed otherwise. It is not intended to limit the scope of the claimed invention. 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 surprising new discovery that Farp2 and Stk25 regulate plasma total lipoprotein and HDL levels, levels that correlate with the onset and progression of CAD. In particular, Farp2 alleles that alter the expression and activity of gene products increase HDL levels. Accordingly, one aspect of the invention provides methods for identifying compounds that alter the expression and / or activity of FARP2, STK25 and related genes and gene products (eg, homologs and orthologs, eg, human orthologs). Another aspect of the invention relates to the use of FARP2, STK25 and its orthologs as biomarkers to monitor the onset, progression or regression of CAD or to assess the efficacy of compounds in the treatment of CAD.

Screening Assay In one aspect, the present invention provides methods for screening (identifying) compounds that increase plasma levels of HDL-C by altering the activity or expression of FARP2, STK25 and homologs and orthologs thereof. Screening may involve, for example, contacting a compound with a biological sample containing a FARP2 or STK25 gene product and monitoring the effect of the compound on the activity of FARP2 or STK25 (or an appropriate homolog or ortholog thereof), or FARP2 or STK25 (or an appropriate Or homologs or orthologs) or by monitoring the effect of the compound on lipoprotein and HDL-C levels in the sample. Farp2 or Stk25 (or an appropriate homolog or ortholog thereof) is in the form of an endogenous or exogenous nucleic acid molecule (eg, an endogenous gene or exogenous vector containing an appropriate reading frame) or a polypeptide or functional fragment thereof obtain.

  The effect of the compound on the modulation of total plasma lipoprotein or HDL-C levels is due to, for example, modulation of the activity of FARP2 or STK25 (or its appropriate homolog or ortholog). Modulation of activity includes, but is not limited to: 1) inhibition or prevention of HDL degradation by the polypeptide or functional fragment thereof, 2) induction of degradation or degradation of the polypeptide or functional fragment thereof, 3) polypeptide Or inactivation of the biological activity of the functional fragment, 4) reduction or inhibition of expression of the nucleic acid molecule, and 5) degradation or destabilization of the nucleic acid molecule.

  If the purpose is to determine the effect of the compound on HDL, parallel samples not receiving the compound are also monitored as controls. The treated and untreated samples are then subjected to, but are not limited to, microscopic analysis, viability testing, replication capability, histology, the level of specific RNA or polypeptide or complex thereof, the level of enzyme activity, and other Compare by appropriate phenotypic criteria, including the ability of cells to interact with cells or compounds. Differences between treated and untreated cells indicate effects that may be attributed to the compound. In one embodiment, the compound is at a rate of at least about 10%, about 20%, about 30%, about 50%, about 70%, about 90%, about 100%, about 150%, about 200% or more. Or inhibition of total plasma lipoprotein or HDL-C level modulation. The screening method comprises: 1) contacting a compound with a first biological sample comprising an appropriate source of HDL and FARP2 or STK25 (or an appropriate homolog or ortholog thereof), measuring the level of HDL in the first biological sample; 3) Determine the level of HDL in the second biological sample, provided that the second biological sample is not exposed to the compound, and 4) Compare the HDL level from 2) with the HDL level from 3) Selecting compounds that are at least about 1.5 times greater.

  In one embodiment, the screening assay involves contacting a cell-free biological sample comprising HDL and an appropriate FARP2 or STK25 (or an appropriate homolog or ortholog thereof) with a compound to modulate total plasma lipoprotein or HDL-C levels. A cell-free assay that measures the ability of a compound to act. Methods for measuring HDL levels are well known in the art (Sugiuchi, et al. Clin. Chem., 41: 717-723, 1995; Izawa et al., J. Med. Pharm. Sci., 37: 1385- 1388, 1997).

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

  In another embodiment, the screening assay is an in vivo screening assay. By performing in vivo screening assays in non-human animals, compounds that effectively inhibit, reduce or delay HDL degradation in animals can be discovered. In one non-limiting example, the compound is administered to a non-human animal at an appropriate dose for an appropriate amount of time, optionally after a high fat diet. The animals are then bled, plasma lipoproteins are isolated, and HDL levels are measured by methods known in the art. When increased HDL levels in animals treated with a compound are compared to HDL levels in animals not treated with a compound, the compound inhibits the activity of, for example, FARP2 or STK25 (or an appropriate homolog or ortholog thereof) Have shown to modulate total plasma lipoprotein and / or HDL-C levels in animals. Preferably, the increase is at least about 1.5 times. Also preferably, the compound has a total plasma lipoprotein and / or HDL-C level in the animal of at least about 10%, about 20%, about 30%, about 50%, about 70%, about 90%, about 100%. , Modulating at a rate of about 150%, about 200% or more.

  If desired, the compound can be subjected to pre-screening by a cell-free screening assay or cell-based screening assay described herein prior to administration to the animal.

  In cell-based screening assays, cells expressing the appropriate FARP2 or STK25 (or appropriate homolog or ortholog thereof) or functional fragment (s) thereof are contacted with the compound and the ability of the compound to modulate activity. Measure. Measuring the ability of a compound to modulate activity can be determined by assessing the biological activity of FARP2 or STK25 (or its appropriate homolog or ortholog) for the appropriate substrate, eg, the catalytic / enzymatic activity, FARP2 or STK25 ( Or a reporter gene comprising a responsive element operably linked to a nucleic acid encoding a detectable marker by assessing its ability to bind to or interact with its appropriate homolog or ortholog) It can be performed by assessing induction or by assessing appropriately regulated cellular responses such as signal transduction or protein / protein interactions. The cell can be a mammalian cell, an insect cell, a bacterial cell, a yeast cell, or the like.

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

Antibodies In one embodiment, the present invention relates to the modulation of HDL nucleic acid and polypeptide levels by using antibodies. Antibodies include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, Fv fragments, F (ab ′) fragments, Fab expression libraries Mention may be made of the fragments produced, anti-idiotype antibodies or other epitope-binding polypeptides. The antibodies of the invention can be monospecific, bispecific, trispecific, or more multispecific. Preferably, antibodies useful in the present invention for detection of mouse ES1 or human CES1 polypeptide are human antibodies or fragments thereof, including scFv, Fab, Fab ′, F (ab ′), Fd, single chain antibodies or Fv. is there. An antibody useful in the present invention may comprise a full heavy or light chain constant region or portion thereof, or may lack it. In one embodiment, an antibody useful in the present invention is a humanized antibody in which the amino acid is replaced with a non-antigen binding region so that it is closely similar to a human antibody, but still retains its original binding ability. obtain. Methods for producing humanized antibodies are known in the art (Teng et al., Proc. Natl. Acad. Sci. 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, the antigen-binding portion of an antibody that binds to a Farp or Stk polypeptide (eg, VH and VL chains) can be “mixed and paired” to create other anti-Farp or Stk binding molecules. The binding of the “mixed and paired” antibodies can be tested using a binding assay (eg, ELISA). When a VH is selected and mixed and paired with a particular VL sequence, typically a VH that is structurally similar to that VH is selected and replaced with a pairing with that VL. Similarly, full length heavy chain sequences from a particular full length heavy chain / full length light chain pair are generally replaced with structurally similar full length heavy chain sequences. Similarly, VL sequences from a particular VH / VL pair are naturally replaced with structurally similar VL sequences. Similarly, full-length light chain sequences from a particular full-length heavy chain / full-length light chain pairing should be replaced by structurally similar full-length light chain sequences. Identification of structural similarity in the above is a process known in the art.

  A human antibody is or is derived from a particular germline sequence if the variable region or full length of the antibody is obtained from a system that uses a human germline immunoglobulin gene as a sequence source. "Includes heavy or light chain variable regions or full length heavy or light chains. In one such system, human antibodies are produced in transgenic mice that carry human immunoglobulin genes. The transgenic is immunized with an antigen of interest (eg, an epitope of a Farp or Stk polypeptide).

  A human antibody that is “product” of or derived from a human germline immunoglobulin sequence compares the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin, and the sequence and sequence of the human antibody It can itself be identified by selecting a human germline immunoglobulin sequence that is very close (ie, with the highest percent identity). A human antibody that is “product” or “derived from” a specific human germline immunoglobulin sequence can be produced by, for example, a naturally occurring somatic mutation or artificially directed mutagenesis, It may include amino acid differences in comparison to the system coding sequence. However, the selected human antibody typically has an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene, and the reproductive species of other species (eg, murine germline sequences). It includes amino acid residues that identify human antibodies as derived from humans when compared with systemic immunoglobulin amino acid sequences. In certain cases, human antibodies have at least 60%, 70%, 80%, 90% or at least 95% or even at least 96%, 97%, 98% or amino acid sequences encoded by germline immunoglobulin genes. It can show 99% amino acid sequence identity.

Camel and dromedaries (Camelus bactrianus), including New World personnel such as llama species (Lama paccos, Lama glama and Lama vicugna) And antibody proteins obtained from members of the Calelus dromaderius family have been characterized with respect to size, structural complexity and antigenicity to human subjects. Certain IgG antibodies found naturally in this mammalian family lack a light chain, and thus have a four-chain quaternary structure with two heavy chains and two light chains typical of antibodies from other animals. Are structurally distinct. See International Publication No. 94/04678.

  A low molecular weight known as “camel nanobody” by generating a region of a camel antibody, a small single variable domain identified as VHH, by genetic engineering technology to produce a small protein with high affinity for the target Antibody-derived protein is obtained. See also US 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 See also: 440-448; Cortez-Retamozo et al., 2002 Int. J. Cancer 89: 456-62; and Lauwereys. Et al., 1998 EMBO J. 17: 3512-3520. Genetically engineered libraries of camel antibodies and antibody fragments are commercially available from, for example, Ablynx (Ghent, Belgium). As in the case of other antibodies derived from non-humans, it is possible to obtain sequences more closely similar to human sequences by modifying the amino acid sequence of camelid antibodies by recombinant techniques, “Humanization” is possible. Thus, the natural low antigenicity of camel antibodies against humans can be further reduced.

  Camel Nanobodies have a molecular weight about one-tenth that of human IgG molecules, and this protein has a physical diameter of only a few nanometers. One consequence of the small size is that camelid nanobodies can bind to antigenic sites that are so small that they do not appear functionally in large antibody proteins, ie camelid nanobodies can be used using classical immunological techniques. Is useful as a reagent for detecting antigens that are normally ambiguous and as a potential therapeutic agent. That is, yet another consequence of the small size may be that camel nanobodies can be inhibited by binding to specific sites in the target protein groove or narrow cleft, and thus more classical than with classical antibodies. It can play a role with a receptivity that closely resembles that of a functional low molecular weight drug.

  In addition, due to its low molecular weight and small size, camel Nanobodies are very heat stable, stable to extreme pH and proteolytic digestion, and poorly antigenic. Another result is that camelid Nanobodies can easily move from the circulatory system into tissues and even pass through the blood brain barrier, thus treating diseases that affect nerve tissue. Nanobodies also facilitate drug transport across the blood brain barrier. See US Patent Publication No. 20040161738 published August 19, 2004. When these features are combined with low antigenicity in humans, great therapeutic potential is expected. Furthermore, these molecules can be well expressed in prokaryotic cells such as E. coli. Camel antibodies are also intended to be included within the scope of the present invention. Thus, one feature of the present invention is a camel antibody or camelid nanobody with high affinity for a Farp or Stk polypeptide.

Diabodies Diabodies are bivalent bispecific molecules in which the VH and VL domains are expressed in a single polypeptide chain, which are too short to allow pairing between two domains in the same chain They are connected by a linker. The VH and VL domains form two antigen-binding sites by pairing with the complementary domains of another chain (eg, Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90: 6444- 6448; Poljak et al., 1994 Structure 2: 1121-1123). Diabodies express two polypeptide chains with the structures VHA-VLB and VHB-VLA (VH-VL configuration) or VLA-VHB and VLB-VHA (VL-VH configuration) in the same cell. Can be manufactured. Most of them can be expressed in soluble form in bacteria.

  Single-chain diabody (scDb) is produced by linking two diabody-forming polypeptide chains with a linker of about 15 amino acid residues (Holliger and Winter, 1997 Cancer Immunol. Immunother., 45 (3- 4): 128-30; see Wu et al., 1996 Immunotechnology, 2 (1): 21-36). scDb can be expressed in bacteria in soluble active monomeric form (Holliger and Winter, 1997 Cancer Immunol. Immunother., 45 (34): 128-30; Wu et al., 1996 Immunotechnology, 2 (1): 21-36 Pluckthun and Pack, 1997 Immunotechnology, 3 (2): 83-105; Ridgway et al., 1996 Protein Eng., 9 (7): 617-21). By fusing a diabody with Fc, a “di-diabody” can be generated (see Lu et al., 2004 J. Biol. Chem., 279 (4): 2856-65).

Genetically engineered and modified antibodies By producing antibodies of the invention using antibodies having one or more VH and / or VL sequences as starting materials, genetically modified antibodies are produced. Thus, the modified antibody may have altered properties from the starting antibody. An antibody is one or more within one or both variable regions (ie, VH and / or VL), eg, within one or more CDR regions and / or within one or more framework regions. Can be engineered by modifying residues. Additionally or alternatively, antibodies can be engineered to modify, for example, the effector function (s) of an antibody by modifying residues in the constant region (s). .

  One type of variable region genetic manipulation that can be performed is CDR grafting technology. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within CDRs show greater diversity among individual antibodies than sequences outside of CDRs. Since CDR sequences are involved in most antibody-antigen interactions, by constructing expression vectors containing CDR sequences from specific natural antibodies grafted from different antibodies with different properties to framework sequences, Recombinant antibodies that mimic the properties of natural antibodies can be expressed (eg, Riechmann et al., 1998 Nature 332: 323-327; Jones et al., 1986 Nature 321: 522-525; Queen et al. al., 1989 Proc. Natl. Acad. See. USA 86: 10029-10033; see U.S. Pat. Nos. 5,225,539 and 5,530,101, 5585089, 569762 and 6180370).

  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 online, www.mrc-cpe.cam.ac.uk/vbase) and Kabat et al. ., 1991 Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication 91-3242; Tomlinson et al., 1992 J. Mol. Biol. 227: 776-798; and Cox et al. , 1994 Eur. J. Immunol. 24: 827-836, the contents of which are incorporated herein by reference.

  VH CDR1, 2, and 3 sequences and VL CDR1, 2, and 3 sequences can be grafted to framework regions having the same sequence as found in the germline immunoglobulin gene from which the framework sequence is derived, or CDR The sequence can be grafted to a framework region containing one or more mutations relative to the germline sequence. For example, in certain cases, it has been found beneficial to introduce mutations to residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (eg, US Pat. 5530101, 5585089, 569762 and 6180370).

  CDRs can also be grafted to framework regions of polypeptides other than immunoglobulin domains. Appropriate scaffolds form a conformationally stable framework in which the graft residues form a localized surface that presents them to bind to the target of interest. For example, CDRs are based on the framework region of fibronectin, ankyrin, lipocalin, neocardinostein, cytochrome b, CP1 zinc finger, PST1, supercoil, LACI-D1, Z domain, or tendramisat. Can be grafted onto a scaffold (see, eg, Nygren and Uhlen, 1997 Current Opinion in Structural Biology, 7, 463-469).

  Another type of variable region modification is the introduction of mutations into amino acid residues within the VH and / or VL CDR1, CDR2 and / or CDR3 regions, thereby known as “affinity maturation”. One or more binding properties (eg, affinity) of an antibody are improved. By performing site-directed mutagenesis or mutagenesis through PCR, mutation (s) can be introduced and the effects on antibody binding or other functional properties of interest are described herein. It can be evaluated by the in vitro or in vivo assay described in the document. Conservative modifications can be introduced. Mutations can be amino acid substitutions, additions or deletions. Furthermore, typically at most 1, 2, 3, 4 or 5 residues in the CDR regions are altered.

  Some genetically engineered antibodies of the invention have modifications made to framework residues within VH and / or VL, for example to improve the properties of the antibody. Typically, the framework modifications are made to reduce the immunogenicity of the antibody. For example, one method is to “back mutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody in which a somatic mutation has been induced 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 framework region sequences to their germline configuration, somatic mutations can be "backmutated" to germline sequences, for example by site-directed mutagenesis or mutagenesis through PCR It is. Such “backmutated” antibodies are also intended to be encompassed by the present invention.

  Another type of framework modification removes a T cell epitope by mutagenizing one or more residues in the framework region or in one or more CDR regions, and the potential of the antibody. Reduce the overall immunogenicity. This method is also referred to as “deimmunization” and is described in detail in US Patent Publication No. 20030153043 by Carr et al.

  In addition to or in place of modifications made within the framework or CDR regions, in the case of the antibodies of the invention, modifications are made in the Fc region by genetic engineering techniques, typically one or more of the antibodies It is also possible to alter functional properties such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cytotoxicity. In addition, the antibody of the invention may be chemically modified (eg, one or more chemical moieties may be attached to the antibody) or its glycosylation altered to re-act one or more functions of the antibody. Modifications can also be made to alter the mechanical properties.

  In one embodiment, the hinge region is modified to alter, ie increase or decrease, the number of cysteine residues in the CH1 hinge region. This method is described in detail in US Pat. No. 5,677,425 by Bodmer et al. Altering the number of cysteine residues in the hinge region of CH1, for example, promotes light and heavy chain association or increases or decreases antibody stability.

  In another embodiment, mutations are introduced into the Fc hinge region of the antibody to reduce the biological half life of the antibody. More specifically, one or more amino acid mutations are performed such that the antibody exhibits reduced SpA binding compared to native Fc-hinge domain staphylococcal (Staphylococcyl) protein A (SpA) binding. Mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment. This method is described in detail in US Pat. No. 6,165,745 by Ward et al.

  In another embodiment, the antibody is modified to increase its biological half life. Various methods are possible. For example, US Pat. No. 6,277,375 reports the following mutations in IgG that increase its in vivo half-life: T252L, T254S, T256F. Alternatively, to increase biological half-life, as described in US Pat. Nos. 5,869,046 and 6121022 by Presta et al., Antibodies are isolated from two loops in the CH2 domain of the Fc region of IgG. It can be modified within the CH1 or CL region to include an extracted salvage receptor binding epitope.

  In yet another embodiment, the effector function of the antibody is altered by altering the Fc region by replacing at least one amino acid residue with a different amino acid residue. 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 whose affinity is altered can be, for example, an Fc receptor or the C1 component of the complement system. This method is reported in detail in US Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

  In another embodiment, one or more selected from amino acid residues, such that C1q binding is altered in the antibody and / or complement dependent cytotoxicity (CDC) is reduced or eliminated. It is also possible to replace multiple amino acids with different amino acid residues. This method is described in detail in US Pat. No. 6,194,551 by Idusogie et al.

  In another embodiment, the ability of the antibody to fix complement is altered by altering one or more amino acid residues. This method is described in detail in WO 94/29351 by Bodmer et al.

  In yet another embodiment, the affinity of the antibody for Fcγ receptors by enhancing the ability of the antibody to transmit antibody-dependent cell-mediated cytotoxicity (ADCC) and / or modifying one or more amino acids. The Fc region is modified to increase This method is described in detail in International Publication No. 00/42072 by Presta. Furthermore, the binding sites in human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have already been mapped, and variants with improved binding have been reported (Shields, RL et al., 2001 J. Biol. Chem. 276: 6591-6604).

  In yet another embodiment, the glycosylation of the antibody is modified. For example, aglycosylated antibodies can be made (ie, antibodies lack glycosylation). Glycosylation can be modified, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, by making one or more amino acid substitutions that result in elimination of one or more variable region framework glycosylation sites, glycosylation at that site can be eliminated. The aglycosylation can increase the affinity of the antibody for the antigen. The above method is reported in more detail in US Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

  Additionally or alternatively, antibodies with altered glycosylation types can be made, such as hypofucosylated antibodies with low amounts of fucosyl residues or antibodies with high bisecting GlcNac structures. The modified glycosylation pattern described above has been demonstrated to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell in which the glycosylation machinery has been altered. Cells with altered glycosylation mechanisms have been reported in the literature and can be used as host cells to produce antibodies with altered glycosylation by expressing the recombinant antibodies of the invention. For example, European Patent No. 1176195 by Hang et al. Relates to a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, so that the antibody expressed in the cell line is fucosyl. It has been reported that the decrease in crystallization. PCT WO 03/035835 by Presta relates to a mutant CHO cell line, Lec13 cells, which has reduced fucose binding capacity to Asn (297) -linked carbohydrate, again expressed in its host cells It has been reported that antibodies exhibit reduced fucosylation (see also Shields, RL et al., 2002 J. Biol. Chem. 277: 26733-26740). WO 99/54342 by Umana et al. Has been genetically engineered to express glycosyltransferases that modify glycoproteins (eg, beta (1,4) -N acetylglucosaminyltransferase III (GnTIII)). As a result, it has been reported that antibodies expressed in genetically engineered cell lines exhibit increased bisecting GlcNac structure, thus increasing the ADCC activity of the antibodies (also Umana et al., 1999 Nat. Biotech. 17: 176-180).

  Another modification of the antibodies herein that is contemplated by the invention is pegylation. PEGylating an antibody can, for example, increase the biological (eg, serum) half life of the antibody. To pegylate an antibody, typically the antibody or fragment thereof is reacted with PEG, eg, PEG, under conditions in which one or more polyethylene glycol (PEG) moieties are attached to the antibody or antibody fragment. React with ester or aldehyde derivative. Pegylation can be performed 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” refers to a PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. All forms are included. In certain embodiments, the PEGylated antibody is an aglycosylated antibody. Protein pegylation methods are known in the art and can also be applied to the antibodies of the present invention. See, for example, European Patent No. 0154316 by Nishimura et al. And European Patent No. 0401384 by Ishikawa et al.

  Furthermore, pegylation can be achieved at any part of the antibody by the introduction of unnatural amino acids. Certain unnatural amino acids are 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 US Pat. No. 7,083,970. Briefly, some of these expression systems introduce nonsense codons such as amber TAG into the reading frame encoding the polypeptide of the present invention by site-directed mutagenesis. The expression vector is then introduced into a host that can utilize a tRNA specific for the introduced nonsense codon and loaded with the selected unnatural amino acid. Particular non-natural amino acids that are useful for the purpose of conjugating such moieties to the polypeptides of the invention are those with acetylene and azide side chains. Polypeptides containing these novel amino acids can then be PEGylated at these selected sites in the protein.

Methods for genetic engineering of antibodies New antibodies by modifying full length heavy and / or light chain sequences, VH and / or VL sequences, or constant region (s) attached thereto, by modifying the antibody Can be made. For example, new genetically engineered antibodies can be made by combining one or more CDR regions of an antibody with known framework regions and / or other CDRs in a genetic recombination procedure. The starting material for the genetic engineering method is one or more VH and / or VL sequences, or one or more CDR regions thereof. It is not necessary to actually produce (ie, express as a protein) an antibody having one or more VH and / or VL sequences, or one or more CDR regions thereof, in order to generate a recombinant antibody. . Rather, by using the information contained in the sequence (s) as a starting material, a “second generation” sequence (s) is generated from the original sequence (s) and then the “second” A “generation” sequence (s) is prepared and expressed as a protein.

  By using standard molecular biology techniques, modified antibody sequences can be prepared and expressed. 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 modified antibody can be assessed by standard assays (eg, ELISA) available in the art and / or described herein.

  In certain embodiments of the antibody genetic engineering methods of the invention, mutations may be introduced randomly or selectively along all or part of the antibody coding sequence. For example, PCT WO 02/092780 by Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or combinations thereof. Alternatively, WO 03/074679 by Lazar et al. Reports on a method for optimizing the physicochemical properties of antibodies by using computer screening methods.

Non-antibody binding molecules Furthermore, the present invention exhibits the functional properties of antibodies, but their frameworks and antigen binding portions are encoded by other polypeptides (eg, encoded by antibody genes or by in vivo recombination of antibody genes). Provided is a binding molecule that is derived from a polypeptide other than the one produced. 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 a global fold similar to that of an antibody variable domain (an “immunoglobulin-like” fold) are suitable scaffold proteins. Scaffold proteins suitable for the production of antigen binding molecules include fibronectin or fibronectin dimers, tenascin, N-cadherin, E-cadherin, ICAM, titin, GCSF-receptor, cytokine receptor, glycosidase inhibitor, antibiotics Chromoprotein, myelin membrane adhesion molecule P0, CD8, CD4, CD2, I class MHC, T cell antigen receptor, I-set domain of CD1, CD2 and VCAM-1, I-set immunoglobulin domain of myosin binding protein C, I-set immunoglobulin domain of myosin binding protein H, I-set immunoglobulin domain of telokin, NCAM, Twitchin, neurologian, growth hormone receptor, erythropoietin receptor, prolactin receptor, interfero - gamma receptor, beta-galactosidase / glucuronidase, beta-glucuronidase, transglutaminase, T-cell antigen receptor, superoxide dismutase, tissue factor domain, cytochrome F, there is a green fluorescent protein, GroEL and thaumatin.

  The antigen binding domain (eg, an immunoglobulin-like fold) of a non-antibody binding molecule can have a molecular weight of less than 10 kD or greater than 7.5 kD (eg, a molecular weight of 7.5-10 kD). The protein used to generate the antigen binding domain is a naturally occurring mammalian protein (eg, a human protein), and the antigen binding domain is no more than 50% (eg, the immunoglobulin-like fold of the protein from which it is derived) , 34% or less, 25%, 20% or 15%). A domain having an immunoglobulin-like fold generally consists of 50-150 amino acids (eg, 40-60 amino acids).

  To generate non-antibody binding molecules, clones with randomized sequences in the regions of scaffold proteins that form the antigen-binding surface (eg, regions similar in location and structure to the CDRs of antibody variable domain immunoglobulin folds) Create a library. Library clones are tested for specific binding to antigens of interest and other functions. By using the selected clone as a basis for further randomization and selection, derivatives with even higher affinity for the antigen can be produced.

  For example, by using the 10th module (10Fn3) of fibronectin III as a scaffold, a high affinity binding molecule is produced. A library is constructed for each of the three CDR-like loops of 10FN3 at residues 23-29, 52-55 and 78-87. To construct each library, DNA segments encoding sequences that overlap each CDR-like region are randomized by oligonucleotide synthesis. Techniques for making selectable 10Fn3 libraries are described in US Pat. Nos. 6,818,418 and 7115396; Roberts and Szostak, 1997 Proc. Natl. Acad. Sci USA 94: 12297; US Pat. No. 6,261,804; et al., described in WO 98/31700.

  By producing the non-antibody binding molecule as a dimer or multimer, the binding activity for the target antigen can be increased. For example, the antigen binding domain is expressed as a fusion with the constant region (Fc) of an antibody that forms an Fc-Fc dimer. See, for example, US Pat. No. 7,115,396.

RNA interference Antisense oligonucleotides are polynucleotides that are complementary to all or part of a target primary transcript (unprocessed transcript) or mRNA and block expression of the target gene (US Pat. No. 5,107,065, International Publication). No. 9928508). The complementarity of the antisense oligonucleotide can be, for example, with a 5 ′ non-coding sequence, a 3 ′ non-coding sequence, or any part of a specific gene transcript at the coding sequence.

  siRNA refers to short interfering RNAs that act to break down mRNA sequences that are homologous to either of the RNA strands in a double helix, and can act on cells such as mammalian cells (including human cells) and bodies such as mammalian bodies. It can induce post-transcriptional silencing of specific genes (including the human body). RNA interference phenomena are well 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 on the sequences and nucleic acids that encode the gene products disclosed herein typically have less than 100 base pairs, and can be, for example, about 30 pb or less, complementary DNA It can be made by methods well known in the art, including the use of chains or synthetic methods. siRNAs can induce interference and can induce post-transcriptional silencing of specific genes in cells such as mammalian cells (including human cells) and bodies such as mammalian bodies (including the human body). A typical siRNA according to the present invention may have an integer number of base pairs of 29 bp or less, 25 bp, 22 bp, 21 bp, 20 bp, 15 bp, 10 bp, 5 bp, before or after or in between. Tools for designing optimal inhibitory siRNA include those available from DNAengine Inc. (Seattle, Washington) and Ambion, Inc. (Austin, Texas).

  Double-stranded ribonucleic acid (dsRNA) molecules can also be used to inhibit expression of a target gene (eg, Farp2 or Stk25) in a cell or mammal, in which case the dsRNA is at least a portion of the mRNA formed by expression of the target gene The complementary region is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and the dsRNA is in contact with the cell expressing the target gene. Sometimes it is intended to inhibit the expression of the target gene by at least 10%, 25% or 40%.

  The dsRNA contains two RNA strands that are sufficiently complementary to hybridize to form a double helix structure. One strand of the dsRNA (antisense strand) is formed during expression of the target gene so that when these two strands are combined under appropriate conditions, they hybridize and form a double helix structure. contains a complementary region from the sequence of the mRNA that is substantially complementary to the target sequence, more generally completely complementary, and the other strand (sense strand) is complementary to the antisense strand. Shall be included. Generally, double helix structures are 15 to 30, more typically 18 to 25, more typically 19 to 24, and most typically 19 to 21 base pairs in length. Similarly, the region of complementarity with the target sequence is 15 to 30, more typically 18 to 25, more typically 19 to 24, and most typically 19 to 21 nucleotides in length. The dsRNA of the present invention may further comprise one or more single-stranded nucleotide overhang (s). dsRNA can be synthesized by standard methods well known in the art through the use of automated DNA synthesizers, such as those commercially available from Biosearch, Applied Biosystems, Inc.

  Those skilled in the art should be familiar with dsRNA containing double-helical structures of 20 to 23, especially 21 base pairs, which have been found to be particularly effective in inducing RNA interference ( Elbashir et al., EMBO 2001, 20: 6877-6888).

  In yet another embodiment, the dsRNA is chemically modified to increase stability. The nucleic acids of the invention are described in well-established methods in the art, such as Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al. (Ed.), John Wiley & Sons, Inc. (New York, New York, USA). Can be synthesized and / or modified by published methods, which are incorporated herein by reference. Chemical modifications include, but are not limited to, 2 ′ modifications, modifications at other sites on the sugar or base of the oligonucleotide, introduction of unnatural bases into the oligonucleotide chain, and ligands or chemical moieties. Examples include substitutions of internucleotide phosphate bonds by covalent bonds and alternative bonds such as thiophosphate groups. Several of the above modifications can be used.

  The chemical coupling of two separate dsRNA strands can be accomplished by any of a variety of known techniques, for example, the introduction of covalent bonds, ionic bonds or hydrogen bonds, hydrophobic interactions, van der Waals or stacking interactions, metal ions It can be achieved by the use of coordination means or purine analogues. In general, chemical groups that can be used to modify dsRNA include, but are not limited to, methylene blue, bifunctional groups, generally bis- (2-chloroethyl) amine, N-acetyl-N ′-(p-glyoxyl). Benzoyl) cystamine, 4-thiouracil and psoralen. In one example, the linker is a hexa-ethylene glycol linker. In this case, dsRNA is produced by solid phase synthesis and a hexa-ethylene glycol linker is incorporated according to standard methods (eg Williams, D.J., and K.B. Hall, Biochem. (1996) 35: 14665-14670). In one embodiment, the 5'-end of the antisense strand and the 3'-end of the sense strand are chemically joined by a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate group. Chemical bonds at the ends of dsRNA are generally formed by triple helix bonds.

  In yet another embodiment, modification of nucleotides in one or both of the two single strands prevents or inhibits the degradation activity of cellular enzymes, such as, but not limited to, certain nucleases. Can be done. Techniques for inhibiting the degradation activity of cellular enzymes on nucleic acids are known in the art and include, but are not limited to, 2′-amino modifications, 2′-amino sugar modifications, 2′-F sugar modifications, There are 2'-F modifications, 2'-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2'-O-methyl modifications and phosphoramidates (eg Wagner, Nat. Med. (1995) 1: 1116). -8). That is, at least one 2'-hydroxyl group of a nucleotide in the dsRNA is replaced with a chemical group, generally a 2'-amino or 2'-methyl group. Alternatively, at least one nucleotide can be modified to form a locked nucleotide. The locked nucleotide contains a methylene bridge that connects the 2'-oxygen of ribose with the 4'-carbon of ribose. Oligonucleotides containing locked nucleotides are described in Koshkin, AA, et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404) It is reported in. Introduction of locked nucleotides into oligonucleotides improves affinity for complementary sequences and increases melting temperatures several degrees (Braasch, DA and DR Corey, Chem. Biol. (2001), 8: 1-7 ).

Use of FARP2, STK25 or homolog / ortholog thereof as a biomarker In one embodiment, the expression level of Farp2 or Stk25 (or an appropriate homolog or ortholog thereof) gene with differential expression is measured in normal and CAD cells and / or tissues To do. In one embodiment, the method for measuring the expression level of a gene (s) comprises one or more of the following steps in any effective order, eg a polynucleotide probe and a biological sample, wherein the probe is the sample: FARP2 or STK25 (or its appropriate homolog or ortholog) in contact with a nucleic acid molecule under conditions effective to specifically hybridize and a FARP2 or STK25 (or its appropriate homolog or ortholog) marker in said sample Detecting the presence or absence of the gene nucleic acid may be included. Also, specific alleles containing clear and related polymorphisms are detected.

  In one embodiment, the probe is applied to samples obtained from both normal and CAD cells and / or tissues, and the presence of Farp2 or Stk25 (or an appropriate homolog or ortholog thereof) nucleic acid molecule is determined by methods well known in the art. To detect. For example, a method for detecting the presence of a marker gene can be performed by an effective method such as Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, and the like.

  In another embodiment, the probe is applied to samples obtained from both normal and CAD cells and / or tissues, and the amount of Farp2 or Stk25 (or an appropriate homolog or ortholog thereof) nucleic acid is determined by methods well known in the art. To detect. The method includes, for example, contacting with a probe, hybridizing, and detecting the hybridized probe, although more quantitative methods and / or comparisons with standards can be used. The amount of hybridization between the probe and target can be measured by appropriate methods such as PCR, RT-PCR, RACE PCR, Northern blot, polynucleotide microarray, Rapid-Scan, etc., quantitative and qualitative Includes both static measurements.

  In another embodiment, FARP2 or STK25 (or an appropriate homolog or ortholog thereof) specific antibody is used to detect FARP2 or STK25 (or an appropriate homolog or ortholog thereof) or its in a biological sample by methods known in the art. The presence of the fragment (s) can be detected. This method includes, to name a few, immunoassays such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, in-gel Competitive and non-competitive assay systems using techniques such as diffusion sedimentation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiation assay, fluorescence immunoassay, protein A immunoassay, etc. Can be included. Such assays are well known in the art (Ausubel et al., 1994, Current Protocols in Molecular Biology, Volume 1, 1, John Wiley & Sons, Inc., New York, which is hereby incorporated by reference). Furthermore, immunoassays useful in the present invention include both homogeneous and heterogeneous methods such as fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA) and nephrometry. Examples include inhibition immunoassay (NIA).

  In another embodiment, the level of FARP2 or STK25 (or an appropriate homolog or ortholog thereof) polypeptide in a biological sample can be measured as a means of monitoring the expression level of FARP2 or STK25 (or an appropriate homolog or ortholog thereof). . Such methods include, for example, obtaining a biological sample, contacting the sample with an antibody specific for FARP2 or STK25 (or an appropriate homolog or ortholog thereof) polypeptide or an appropriate epitope thereof, and forming an immune complex with the antibody. The amount of immune complex formation is indicative of the level of FARP2 or STK25 (or an appropriate homolog or ortholog thereof) polypeptide. FARP2 or STK25 (or an appropriate homolog or ortholog thereof) polypeptide level in a biological sample obtained from a subject afflicted with CAD was obtained in advance or subsequently from a biological sample taken from a normal subject or from the same subject. This method is beneficial when compared to the level of FARP2 or STK25 (or its appropriate homolog or ortholog) in one or more samples.

  Also, measurement of the amount of FARP2 or STK25 (or its appropriate homolog or ortholog) polypeptide may correlate with the progression of CAD. FARP2 or STK25 (or its appropriate homolog or ortholog) polypeptide levels can be used predictively to assess whether a biological sample contains cells that are prone to CAD disease or a specific treatment plan Can be used to stand up.

Diagnostic Assays Measuring a detectable increase or decrease in the expression level of FARP2 or STK25 (or an appropriate homolog or ortholog thereof) in a subject with CAD compared to a normal subject is beneficial for the disease state and / or treatment Provide a means of diagnosing or monitoring the response. Accordingly, the present invention provides a method for detecting CAD or atherosclerosis or, alternatively, a method for determining whether a subject is at risk of developing CAD or atherosclerosis.

  For clinical applications, human tissue samples are analyzed for the presence and / or absence of the appropriate Farp2 or Stk25 (or its appropriate homolog or ortholog) encoding nucleic acid and / or FARP2 or STK25 (or its appropriate homolog or ortholog) polypeptide. Can be screened. The sample can include a tissue sample, whole cell, cell lysate, or isolated nucleic acid, including, for example, a puncture biopsy core, a surgically excised specimen, lymph node tissue, plasma or serum. In certain embodiments, nucleic acids extracted from these samples can be amplified using techniques known in the art. The level of detected FARP2 or STK25 (or its appropriate homolog or ortholog) and / or plasma lipoprotein or HDL-C is compared to that in a normal tissue sample.

  In one embodiment, the diagnostic method comprises determining whether the subject is suffering from CAD by detecting the mRNA, cDNA or polypeptide level of Farp2 or Stk25 (or an appropriate homolog or ortholog thereof). . A significant change in the level of expression of activity in a subject compared to that in a healthy normal subject is an indication of CAD or susceptibility to 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 more.

  Alternatively, diagnostic methods can be performed by detecting FARP2 or STK25 (or an appropriate homolog or ortholog thereof) polypeptide or functional fragment thereof using an antibody. In one embodiment, the method comprises a comparison of the level of an appropriate FARP2 or STK25 (or its appropriate homolog or ortholog) polypeptide molecule in a biological sample from a subject with a control level of the polypeptide molecule, In some cases, a significant change in FARP2 or STK25 (or its appropriate homolog or ortholog) polypeptide level is indicative of CAD in the subject. The term “significant change” refers to at least about 10%, about 20%, about 25%, about 30%, preferably about the amount of polypeptide or functional fragment thereof compared to a biological sample from a healthy normal subject. Refers to a change of 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 more.

Prognosis, staging and monitoring of coronary artery disease In one aspect, the present invention provides CAD based on testing expression levels of Farp2 or Stk25 (or appropriate homologs or orthologs thereof) nucleic acids, polypeptides and / or functional fragments thereof. Provides a method for determining the onset, prognosis, and stage of disease. As used herein, prognosis refers to a probable course of disease and prediction of outcome.

  In general, the method used for CAD prognosis or staging involves comparing the amount of the appropriate Farp2 or Stk25 (or its appropriate homolog or ortholog) in the sample of interest with that of the control sample. To detect the relative difference in the expression level of Farp2 or Stk25 (or its appropriate homolog or ortholog). This difference can be measured qualitatively and / or quantitatively. A control sample can be a non-CAD or normal sample, a sample that is known not to progress, or a sample that is known to be progressing.

  As used herein, CAD stage (classification) refers to the degree of progression of events in which CAD occurs and induces symptoms. In addition, staging is a process used to describe how a CAD pathology is progressing in a patient. The method of the present invention is useful for testing CAD staging. Staging can be accomplished by measuring the expression level of the appropriate Farp2 or Stk25 (or its appropriate homolog or ortholog) relative to the control level. The reference level can be from a healthy sample, not from CAD, or from a CAD sample of a different stage in disease development.

  Furthermore, the present invention provides a method for monitoring the progression or recurrence of CAD in a subject by measuring the expression level of a suitable Farp2 or Stk25 (or suitable homolog or ortholog thereof) nucleic acid or polypeptide or functional fragment thereof over time. I will provide a.

  In one embodiment, the method comprises: a) measuring the expression level of an appropriate Farp2 or Stk25 (or appropriate homolog or ortholog thereof) nucleic acid molecule in a subject at an initial time point, and b) Farp2 or Stk25 in a subject at a subsequent time point. Measuring the expression of the nucleic acid (or its appropriate homolog or ortholog), and c) comparing the expression level at the first time point with that at the subsequent time point, in which case a significant change in the expression level is Use as an indicator of onset, progression or regression.

  In another embodiment, the method comprises: a) measuring the expression level of FARP2 or STK25 (or an appropriate homolog or ortholog thereof) polypeptide molecule in the subject at the first time point; and b) FARP2 or STK25 in the subject at the subsequent time point. Measuring the expression of the polypeptide (or an appropriate homolog or ortholog thereof) and c) comparing the expression level at the first time point to that at the subsequent time point, wherein the significant change in expression level is Use as an indicator of onset, progression or regression.

  An increase in the expression level of Farp2 or Stk25 (or an appropriate homolog or ortholog thereof) nucleic acid or polypeptide or functional fragment thereof at a subsequent time point relative to the initial time point indicates that the disease has progressed to a more severe stage. . In contrast, a decrease in expression levels at subsequent time points indicates that the disease has transitioned to a less severe stage.

Therapeutic and therapeutic compound efficacy In another aspect, the present invention also provides a method that allows for the evaluation and / or monitoring of patients who appear to be successful in both traditional and non-traditional treatments and therapies for CAD. provide. The advantage of the present invention is that the patient can follow any course of treatment (s) by monitoring and screening patients over time who may benefit from one or several of the available treatments over time. Or whether it is justified to change, modify or discontinue treatment, or whether the patient's medical condition or stage has progressed.

  By identifying the correct patient for a particular therapy according to the present invention, the efficacy of the treatment can be increased, and exposure of the patient to unwanted life-threatening side effects of the therapy can be avoided. The ability to monitor a patient undergoing a course of treatment using the method of the present invention can determine whether the patient is responding appropriately to treatment over time, modifying the dosage or dosage or delivery method Alternatively, it can be determined whether adjustments should be made and it can be determined whether the patient is on the way to treatment, is returning, or has entered a more severe or advanced stage.

  The monitoring method according to the present invention allows a patient's body fluid sample to be tested or analyzed for a period of time, for example during treatment or therapy, or during the course of a patient's illness. Reflect serial or continuous test or analysis results. For example, in a series test, the appropriate Farp2 or Stk25 in a patient is determined by measuring FARP2 or STK25 (or its appropriate homolog or ortholog) level during the course of treatment and / or during the course of the disease, according to the method of the invention. For the purpose of observing, checking or examining the expression level of a nucleic acid or polypeptide or functional fragment thereof (or an appropriate homolog or ortholog thereof), the same patient provides a body fluid sample, such as serum or plasma, or the same patient A sample has been taken.

  Similarly, FARP2 or STK25 (or an appropriate homolog or ortholog thereof) in a biological sample for the purpose of measuring the efficacy, response and response to a male or female disease state and / or treatment or therapy that a male or female is undergoing. Patients can be screened over time to assess levels. By taking one or more biological samples optimally from the patient prior to the treatment or treatment process or at the beginning of the treatment or treatment, the patient is treated and undergoes clinical and medical evaluation. It may be desirable to supplement the analysis and evaluation of patient progression and / or response at one or more subsequent time points during a period of time.

  Levels can be monitored over a period of days, weeks, months, years, or at various intervals. Patient fluid samples, such as serum or plasma samples, are collected at intervals determined by an expert such as a physician or clinical researcher, and FARP2 or STK25 (or its appropriate homolog or Ortholog), lipoprotein or HDL levels are measured and compared to levels in normal individuals. For example, in accordance with the present invention, patient samples may be taken and monitored monthly, every two months, or any combination of 1, 2 or 3 month intervals. It is advisable to monitor patient samples quarterly or more frequently. Treatment or disease progression or outcome is measured by comparing the level found in the patient to the level in normal individuals, the patient's own level obtained from a previous examination period.

  Appropriate Farp2 or Stk25 (or its appropriate homolog or ortholog) level, preferably to a level found in normal individuals, to a level around, or below it, indicating a decrease in total plasma lipoprotein and an increase in plasma HDL-C The decrease over time is indicative of treatment progress or efficacy, and / or disease improvement, remission, and the like.

Kits The present invention also provides kits comprising reagents necessary for the detection of the expression level (nucleic acid or polypeptide level) of the appropriate Farp2 or Stk25 (or its appropriate homolog or ortholog) in a biological sample. Reagents can include the specific probes / primers and antibodies described above. The kit may also include a control / standard value or a series of controls / standard values that indicate normal conditions and various clinical progression stages of the disease. In a preferred embodiment, the control / standard value or series of controls / standard values is indicative of normal status and various clinical progression stages of CAD. In addition, the kit may include a positive control and / or a negative control for comparison with the test sample. Negative controls can include samples that do not have Farp2 or Stk25 (or an appropriate homolog or ortholog thereof) nucleic acid or polypeptide. A positive control may comprise a sample with various known levels of the appropriate Farp2 or Stk25 (or its appropriate homolog or ortholog) nucleic acid or polypeptide. The kit can also include instructions for performing the assay and interpreting the results. In the case of an antibody-based kit, the kit is bound to a first antibody (eg, a solid support) that binds to, for example, (1) FARP2 or STK25 (or an appropriate homolog or ortholog thereof) polypeptide or functional fragment thereof. ) And optionally (2) a FARP2 or STK25 (or appropriate homolog or ortholog thereof) polypeptide, an epitope thereof or a first antibody, and a second different antibody conjugated to a detectable label. In the case of an oligonucleotide-based kit, the kit can be, for example, (1) an oligonucleotide that hybridizes to an appropriate Farp2 or Stk25 (or its appropriate homolog or ortholog) nucleic acid sequence, eg, a detectably labeled oligo A pair of primers useful for the amplification of nucleotides or (2) Farp2 or Stk25 (or an appropriate homolog or ortholog thereof) nucleic acid molecule may be included. The kit can also include, for example, a buffer, a preservative, or a protein stabilizer. In addition, the kit can include components necessary for the detection of a detectable label (eg, an enzyme or a substrate). The kit can also include a control sample or a series of control samples that can be subjected to the assay and compared to the test sample. Each component of the kit can be enclosed in a separate container, and the various containers can all be contained in a single package with instructions for interpreting the results of the assay performed using the kit.

  The kit can be used to determine whether a subject has or is at increased risk for developing CAD or atherosclerosis. Further, the kit can be used to measure, stage, or monitor the progression of CAD or atherosclerosis prognosis. Furthermore, the kit can be used for drug screening or selection of treatment for CAD or atherosclerosis.

Pharmaceutical Compositions In another aspect, the invention provides an HDL modulating agent (eg, monoclonal antibody or antigen-binding portion (s)), antisense, of the invention formulated with a pharmaceutically acceptable carrier. A composition, for example a pharmaceutical composition, comprising one or a combination of siRNA, low molecular weight molecules) is provided. The composition may comprise one or a combination of binding molecules (eg, two or more different). 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.

  The pharmaceutical compositions of the invention can also be administered in combination therapy, ie in combination with other agents. For example, the combination therapy can include an HDL modulating agent in combination with at least one other cholesterol-lowering agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail in the section relating to the use of the agents of the invention below.

  As used herein, "pharmaceutically acceptable carrier" includes all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. To do. The carrier is of course suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound may be coated with a substance that protects the compound from the action of acids and other natural conditions that may inactivate the compound.

  The pharmaceutical composition of the present invention may comprise one or more pharmaceutically acceptable salts. “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not cause undesired toxicological effects (eg, Berge, SM, et al., 1977 J Pharm. Sci. 66: 1-19). Examples of the salts include acid addition salts and base addition salts. Acid addition salts include non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid and the like, and non-toxic organic acids such as aliphatic mono- and di- Some are derived from carboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include alkaline earth metals such as sodium, potassium, magnesium, calcium, and non-toxic organic amines such as N, N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, Some are derived from diethanolamine, ethylenediamine, procaine, and the like.

  The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and other oil-soluble antioxidants such as ascorbyl palmitate. , Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like, and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and so on.

  Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (eg, glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil And injectable organic esters such as ethyl oleate. The proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

  These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be reliably achieved both by sterilization, the above, and the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, absorption of injectable pharmaceutical forms can be extended by the inclusion of agents delaying 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 drugs for pharmaceutically active substances is known in the art. Except insofar as conventional media or drugs are not compatible with the active compounds, their use in the pharmaceutical compositions of the present invention is also contemplated. Supplementary active compounds can also be incorporated into the compositions.

  The therapeutic composition 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, polyols (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 material 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, isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride may be included in the composition. It is also possible to extend absorption of injectable compositions, for example by including in the composition an absorption delaying agent, such as monostearate salts and gelatin.

  Sterile injectable solutions can be prepared by mixing the required amount of the active compound in a suitable solvent with one or a combination of the above-listed ingredients, and then sterile microfiltration if necessary. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients selected from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the process is vacuum drying and freeze drying (freeze drying) in which the active ingredient and the desired additional ingredient powder are produced from the pre-sterilized filtered solution. .

  The amount of active ingredient that can be combined with the carrier materials 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 that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that provides a therapeutic effect. Generally, out of 100 percent, this amount is about 0.01 percent to about 99 percent active ingredient, about 0.1 percent to about 70 percent, or about 1 percent in combination with a pharmaceutically acceptable carrier. The active ingredient in a proportion of about 30 percent.

  Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, it may be administered in several divided doses over time, or the dose may be increased or decreased as appropriate according to the urgency of the treatment situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to a physical discrete unit suitable as a unit dose for the subject being 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 description of the unit dosage forms of the present invention is defined by the limitations inherent in the active compound formulation techniques relating to the unique characteristics of the active compound and the specific therapeutic effect to be achieved and the treatment depending on the sensitivity in the individual. It depends.

  For administration of HDL modulating agents such as antibodies, the dosage ranges from about 0.0001 to 100 mg / kg, more usually 0.01 to 5 mg / kg, based on the body weight of the host. For example, the dosage is in the range of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg or 10 mg / kg or 1-10 mg / kg based on body weight. Exemplary treatment regimens are once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every three months, or every 3-6 months One dose is required. Dosing regimens for the antibody include 1 mg / kg (body weight) or 3 mg / kg (body weight) by intravenous administration, and the antibody is administered using one of the following dosing schedules: every 4 weeks for 6 doses, then Every 3 months; every 3 weeks; 3 mg / kg (body weight) once, then every 3 weeks, 1 mg / kg (body weight).

  Depending on the method, two or more binding molecules (eg, monoclonal antibodies) with different binding specificities may be administered simultaneously, with the dose of each antibody being administered within the indicated range. To do. HDL modulating agents are usually administered frequently. The interval between single doses can be, for example, every week, every month, every three months, or every year. Depending on the blood level measurement of the HDL modulating agent (eg, antibody) in the patient, irregular intervals may be indicated. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml and in some methods about 25-300 μg / ml.

  Alternatively, the HDL modulating agent can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the HDL modulating agent in the patient. For example, in the case of antibodies, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic applications, a relatively low dose is administered at relatively infrequent intervals over an extended period of time. Some patients may continue to receive lifelong treatment. For therapeutic applications, relatively high doses may be required at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement in disease symptoms . Thereafter, the patient can be administered a prophylactic dosing program.

  The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is such that the effective dose is effective to achieve the desired therapeutic response for the particular patient, composition and mode of administration without causing toxicity to the patient. It can be modified to obtain the components. The selected dosage level depends on the particular composition of the invention used, or the activity of its ester, salt or amide, route of administration, administration time, excretion rate of the particular compound being used, duration of treatment, Other drugs, compounds and / or substances used in combination with the particular composition used, the age, sex, weight, medical condition, general health and history of the patient being treated and similar well known in the medical community It varies according to various pharmacokinetic factors including factors.

  A “therapeutically effective amount” of an HDL-modulating agent is a decrease in the severity of a disease symptom (eg, a decrease in plasma cholesterol or a decrease in symptoms of a cholesterol-related disease), an increase in the frequency and duration of asymptomatic periods of the disease, or It is intended to prevent defects or disability caused by sickness.

  The composition can be administered by one or more routes of administration using one or more of a variety of methods known in the art. One skilled in the art will recognize that the route of administration and / or method will vary depending on the desired result. The route of administration of the HDL modulating agent includes, but is not limited to, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. As used herein, the expression “parenteral administration” refers to a mode of administration by normal injection other than enteral and topical administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal. Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrastemal injection and infusion Is included.

  Alternatively, the HDL modulating agent can be administered by a parenteral 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 as controlled release formulations, including, for example, implants, transdermal patches, and microencapsulated delivery systems using carriers that will protect the compound against rapid release. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polybutyric acid. Many methods for producing the above formulations are patented or known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by J.R. Robinson, Marcel Dekker, Inc., New York (1978).

  The therapeutic composition can be administered by medical devices well known in the art. For example, in one embodiment, the therapeutic composition of the present invention can be administered by a needleless hypodermic injection device, such as the device shown in US Pat. Examples of known implants and modules useful in the present invention include U.S. Pat. No. 4,487,603, which shows an implantable microinfusion pump that dispenses drugs at a controlled rate, U.S. Pat. U.S. Pat. No. 4,486,194, U.S. Pat. No. 4,447,233 showing a drug infusion pump delivering drug at a precise infusion rate, U.S. Pat. No. 4,447,224 showing variable flowable implantable infusion device for continuous drug delivery, multi-chamber US Pat. No. 4,439,196, which shows an osmotic drug delivery system with compartments, and US Pat. No. 4,475,196, which shows an osmotic drug delivery system. These patents are incorporated by reference. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

  In certain embodiments, the HDL modulating agent can be formulated to ensure proper distribution in vivo. For example, the blood brain barrier (BBB) eliminates many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention exceed the BBB (if desired), they can be formulated, for example, in liposomes. See, for example, US Pat. Nos. 4,522,811, 5,374,548 and 5,399,331 for methods of producing liposomes. Liposomes contain one or more moieties that are selectively transported to specific cells or organs, which can drive targeted drug delivery (see, eg, VV Ranade, 1989 J. Cline Pharmacol. 29: 685). . Examples of targeting moieties include folate or biotin (see, eg, Low et al., US Pat. No. 5,416,016), mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun. 153: 1038), antibodies (PG Bloeman et al., 1995 FEBS Lett. 357: 140; M. Owais et al., 1995 Antimicrob. Agents Chernother. 39: 180), surfactant protein A receptor (Briscoe et al., 1995 Am. J Physiol. 1233: 134), p120 (chreier et al., 1994 J. Biol. Chem. 269: 9090). See also K. Keinanen; M.L. Laukkanen, 1994 FEBS Lett. 346: 123; J.J. Killion; I.J. Fidler, 1994 Immunomethods 4: 273.

Typical Example Example 1
There is strong evidence that elevated functional HDL plasma levels reduce the risk of coronary artery disease (Gotto, A. et al., J. Am. Coll. Cardiol., 43: 717-724, 2004). ). Apoa2 was previously identified as the Chr1 QTL gene responsible for many but not all of the hybrids (Wang, X. et al., Genome Res., 14, 1767-1772, 2004). Soat1 was identified as the most likely QTL gene for four additional hybrids; Cast × 129, B6 × KK, Pera × DBA / 2 and RIII × 129. However, the following information indicated that there could be a third QTL in Chr1. First, the wide confidence interval for the C57BL / 6 (B6) and 129S1 / SvImJ (129) hybrids indicated the presence of two closely linked QTLs, Hdlq14 and Hdlq15 (Ishimori, N. et al., Arterioscler. Thromb. Vasc. Biol., 24: 161-166, 2004). Apoa2 is a QTL gene for Hdlq15, but Soat1 shows no expression or sequence differences between B6 and 129 strains, so Soat1 is a causative gene for Hdlq14 even though it is located in the Hdlq14 region Unexpectedly, it shows that a third QTL and a third QTL gene may be present. Second, the human HDL QTL in Chr 2q36.1-37.1 (FIG. 1) shows homology with this region, but does not contain Apoa2 or Soat1, and also in this respect the third QTL is , Suggesting that it may be present in the mouse Hdlq14 region that is homologous to human 2q QTL.

  To confirm Hdlq14 in chromosome (Chr) 1 identified in hybrid B6 × 129 and to identify the QTL gene, Apoa2 in Chr1 is the same so as not to have a strong effect on nearby QTL, NZB and Quantitative trait locus (QTL) analysis was performed on the F2 population formed from the NZW line. Hdlq14 was verified and the decision interval was reduced from 45 Mb with 225 genes to a region containing only two genes: Farp2 and Stk25. In genetically diverse 43 mouse strains, strains with the FARP2 Leu821 variant were found to have significantly higher plasma HDL levels than those with the FARP2 Pro821 variant.

  QTL for HDL in Chr1 is complex. In 23 hybrids published using HDL as phenotype, 15 of them detected QTL in Chr1 (Rollins, J. et al., Trends Cardiovasc. Med., 16: 220-234, 2006). ). The extensive and complex LOD score plot for many of these Chr1 QTLs suggests a large number of QTLs. Statistical evidence for at least two QTLs, namely Hdlq14 and Hdlq15, was obtained in one hybrid B6 × 129 (Ishimori, N. et al., Arterioscler. Thromb. Vasc. Biosc., 24: 161-166, 2004). ). Hdlq15 having a peak at 104 cM is determined by an amino acid change from Ala61 to Val61 in the Apoa2 gene. By sequencing, inbred lines have 5 different Apoa2 genes, with 9 amino acid changes between them, but only the hybrids where QTL was detected differed between alanine or valine at position 61 Indicated. Since Apoa2 QTL has a strong effect, it can only include the strong influence of nearby QTL. Examination of the cross with QTL in Chr1, which was not explained by the Apoa2 polymorphism, showed that Soat1 is a QTL gene in four hybrids. However, since the strains examined did not differ in the coding region sequence or expression level of Soat1, Soat1 was detected in a B6 × 129 hybrid and could not be a QTL gene for Hdlq14 located in cM80.

  Using a fine SNP map from Broad Institute (see their website, broad.mit.edu/snp/mouse/), a line was selected to obtain a hybrid with the following characteristics: (1) Line Have the same haplotype in the Soat1 gene, so there is no QTL in Soat1, (2) the strain has the same amino acid at position 61 in the Apoa2 gene, so there is no QTL in Apoa2, and (3) a different haplotype in the Hdlq14 region The presence of. Only a pair of readily available strains meet these criteria. NZW / LacJ (NZW) and NZB / BINJ (NZB) have the same haplotype in the Soat1 gene (data not shown), and both have a valine at position 61 in Apoa2. Over most, but not all, of the 82-162 Mb region defined by the Hdlq14 confidence interval, NZW had the same haplotype as 129 and NZB had the same haplotype as B6. For the 82-162 Mb region, the NZW haplotype is the same as NZB. If the QTL gene responsible for Hdlq14 is located in those regions that appear to be identical due to pedigree between NZW and NZB, QTL is not detected. However, if the QTL gene is located in a region where NZB and NZW are different, we can of course map this QTL without the interference of differences induced by the Apoa2 and Soat1 genes.

  Hdlq14 is confirmed in a hybrid between NZB and NZW. In 264 (NZB × NZW) F2 male and female mice, plasma HDL levels were measured after a high fat diet for 8 weeks. The log-transformed HDL distribution in F2 progeny was almost normal (FIG. 2A). Genes affecting plasma HDL with a peak near D1Mit336 (58.7 cM, 98.44 Mb in NCBI Build 36) and a marked LOD score of 3.02 (FIG. 2B, line I) by interval mapping for Chr1 The seat became clear. The 95% confidence interval defined by 1 unit decrease in LOD score on one side of the peak marker is 80-125 Mb, overlapping the 82-162 Mb Hdlq14 region identified in the 129 × B6 hybrid. F2 mice that are NZW homozygous at D1Mit336 had significantly higher HDL levels compared to mice with a heterozygous or homozygous NZB genotype at this locus (FIG. 2C), so the allele for high HDL is recessive Met.

  By using a bioinformatics tool, the Hdlq14 region is narrowed to 19 genes. The confidence intervals for QTL identified from these hybrids are very large: 80 Mb with the 487 gene for the B6 × 129 hybrid and 45 Mb with the 225 gene for the NZB × NZW hybrid (FIG. 3A, respectively). And 3B). In order to narrow the QTL region and narrow the number of candidate genes, we used various bioinformatics and statistical tools. These tools, including a combination of mating, comparative genomics and haplotype analysis, allow a gradual narrowing of the QTL interval that determines the priority of candidate genes for further analysis.

  Hdlq14 is narrowed to 27.3 Mb containing 125 genes by combining mating. The large confidence interval for a typical QTL is due to a limited number of recombination events. Combining crosses increases the number of recombination and reduces the QTL interval (Li, R. et al., Genetics, 169: 1699-1709, 2005). Raw data for Hdlq14 from both the current (NZB × NZW) F2 hybrid and the original (B6 × 129) F2 hybrid, B6 and NZB genotypes as single low HDL alleles, single high HDL alleles As 129 and NZW genotypes by recoding. Reanalysis of the combined chromosome 1 data increased the LOD score for Hdlq14 to 5.01 (FIG. 2B, line II) and the 95% confidence interval was narrowed to 27.3 Mb, ranging from Mb 89.8 to 117.1 Mb. (FIGS. 3C and 2B, line II), the number of genes was reduced to 125.

  Hdlq14 is narrowed to 6.2 Mb containing 89 genes by comparative genomics. Rodent and human QTL for the same trait are mapped to homologous genomic locations. Because the mammalian genome is degraded and rearranged during evolution, there are approximately 340 homologous segments between mouse and human (LA., P., Mamm. Genome, 14: 429-436, 2003). . Thus, as much as 20 cM of each mouse QTL can be homologous with 3-4 different human chromosomes. By comparing these homology maps, assuming that homology QTL is caused by the same gene in both species, the size of QTL in both species can be reduced. This assumption has been found to be true for a number of traits initially identified in mice 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). HDL QTL identified in human Chr 2q37.2-37.3 was compared to Hdlq14 by using genomic homology between mouse and human. This human QTL shows homology only for the 84-96 Mb portion of Hdlq14. The use of comparative genomics narrows the distal end of QTL and eliminates the 96-117 Mb region, since the crossing combination has already eliminated a region close to 89.8 Mb. This step narrows the QTL region to 6.2 Mb and 89 genes (FIG. 3D).

  Hdlq14 is narrowed down to 2.3 Mb containing 19 genes by haplotype analysis. Haplotypes from all four parental lines are shared between NZW and 129 lines by comparing them throughout the reduced QTL interval (Mb89.8-96), but differ between NZB and B6 lines. The region was identified. A common haplotype block was defined as 3 or more contiguous shared alleles as illustrated in FIG. 4 (only partial analysis of the region is shown). In defining regions that could contain genes, SNPs above and below the endpoint were used (eg, 90.35 Mb to 90.51 Mb in FIG. 4). SNP maps from the Broad Institute with 439 SNPs with an average density of 1 SNP / 14.3 kb in the 89.8-96 Mb region were first used and have many additional SNPs, but one of the 4 parental lines , NZB was confirmed in the Perlegen database not included. By haplotype analysis, the QTL region was narrowed to 2.3 Mb containing only 19 genes (FIG. 3E, Table 1).

  Evaluation of reduced candidate list for expression differences. To assess whether any of the genes show differential expression between parental lines, we compared transcription profiles using an Affymetrix expression microarray. These data were obtained from another experiment (during manuscript preparation) comparing liver expression in 12 males and females fed a normal or high fat diet. Three of the four parent lines of these hybrids; 129, B6 and NZB lines were included in the 12-line survey. Of the 15 candidate genes tested, all but two Riken clones had probes on the Affymetrix chip. Only probes that accurately blast to the only position were examined. Two of these 13 genes, Farp2 and Stk25, showed significant expression differences between B6 and 129 lines (crossbred parents). Since microarray experiments do not include NZW, comparisons between NZW and NZB are not possible. However, haplotype data showed that NZW naturally has the same regulatory information as 129, so NZB was compared to 129. The differential expression remained for Stk25 but not Farp2. Since B6 and NZB both have the same haplotype for Hdlq14 (Table 2), neither gene shows differential expression between B6 and NZB as expected.

  Evaluation of candidates for sequence differences. QTL may be due to differences between parental strains in protein expression or function. The functional difference is due to sequence differences in the coding region. To assess whether any of these genes have a polymorphism in the coding region, we examined the SNPs of the Mouse Phenome Database on a gene-by-gene basis for coding differences between strains. These are listed in Table 3. For 8 genes with coding region mutations, the sequence changes are classified according to whether the base change results in the same amino acid (coding region-Cs for synonyms) or changed amino acid (coding-Cn for non-synonymous) did. Although it is expected that amino acids need to be changed to induce functional changes in proteins, it has been established that synonymous changes can affect protein levels and function if codon frequencies are changed. If the rate of protein synthesis induced by rare codons is slow, not only will the protein level change, but the function may also be changed by causing abnormal folding and secondary structure. Each synonymous change was evaluated using a codon frequency table (available on the website kazusa.or.jp/codon). Only two induced changes in codon frequency. In the E030010N08Rik gene, the frequency of codons used was changed from 14% (GTT) to 23% (GTC). In the Stk25 gene, the frequency of codons used was changed from 41% (ACA) to 8% (ACG) (Table 3).

  Each nonsynonymous amino acid change in 6 genes was also evaluated (Table 3). None of the genes had changes that affected polarity, and only Glrp1 had changes that affected the charge of the protein. Changes in cysteine found in the Mterfd2 gene could change important disulfide bonds, and changes in proline found in the Farp2 gene could affect protein folding, ie its function.

  The SNP database does not contain all SNPs, even from Perlegen resequencing. Therefore, sequencing of five of these genes provided confirmation that all possible polymorphisms were found; its name, HDL binding protein, is a certain HDL gene Hdlbp, as well as having non-synonymous amino acid changes, respectively, since it suggests that it may have a role, Sned1, Mterfd2, Pask and Farp2. At least 50 nucleotides of each exon and adjacent intron were sequenced for each gene. Six genes were identified as having polymorphisms with altered amino acids (Table 3).

  Problems have also been raised as to whether the amino acids changed in any of these genes are in conserved regions or known functional domains. Amino acid changes in SNED1, MTERFD2 and PASK did not occur in conserved regions or known functional domains. The gene Glrp1 was found only in mice and showed no homology. The FARP2 mutant Pro821Leu (FIG. 5A) was found in a region conserved among mammalian species (FIG. 5B), which contains phospholipases, GTPase regulatory proteins and protein kinases, and lipids. Identified in proteins with diverse enzymatic or regulatory functions, such as binding proteins (Blomberg, N. et al., Trends Biochem Sci., 24: 441-445, 2007) as plextrin homology (PH) domains Recognized (Lemmon, M., Biochemical Society symposium, 81-93, 2007). The plextrin domain itself is thought to bind lipids (Klopfenstein, D. & Vale, R., Mol. Biol. Cell, 15: 3729-3739, 2004). Since Farp2 was a promising candidate, the genes were sequenced in 12 lines (129, A / J, B6, C3H, CAST, DBA2, FVB, NOD, NZB, NZW, RIII, SM and SJL) and functional SNPs (Rs13475988) was determined in four additional lines (Pera, I / LnJ, NZO and NON).

  Evidence from previous hybrids points to Farp2 and Stk25 as QTL genes. By sequence and expression assessment, the number of candidate genes is likely to be two candidates (expression differences and amino acid changes (Leu821Pro) in conserved functional domains with large differences in codon usage (ACA to ACG) and conserved functional domains) Farp2) and several other genes that could not be completely excluded (two Riken clones that were not tested for expression, Sned1, Mterfd2 and Pask with amino acid changes in the non-conserved region, and one amino acid change , Glrp1), a protein that is not shared with other mammalian species.

  The hybrid NZO × NON was examined. These parent lines have different haplotypes in the Riken clone and the region containing the Sned1, Mterfd2, Pask and Glrp1 genes, but have the same haplotype for Farp2 and Stk25 (FIG. 6B). Since this hybrid does not have a QTL in the Hdlq14 region (FIG. 6A), this hybrid does not have a different QTL gene. This eliminates the genes at the top of the region and reduces the list of candidate genes to only those in regions with the same haplotype; Farp2 and Stk25. In order to confirm these findings, the hybrid B6 × C3H was examined. This hybrid has the same haplotype in the Farp2 and Stk25 regions (FIG. 6B). When one of these two genes is a QTL gene, naturally there is no QTL in cM55.3. The difference in Soat1 between B6 and C3H and the difference in Apoa2 for both hybrids resulted in the presence of QTL in cM81.6 and 92.6, but the LOD score plot from this hybrid (FIG. 6A) is shown in cM58. Indicates that there is no QTL. Both sets of evidence are inferred from the lack of QTL, and it is always difficult to be confident about negative data. The final mating test provides positive evidence and can further confirm this finding, possibly indicating the actual functional polymorphism. Chr1 plots from hybrid x DBA / 2 were compared. The PERA × DBA / 2 hybrid has an additional QTL for cM55 on Chr1 (FIG. 6A), and a comparison of these two haplotypes shows that they differ for SNA in the Farp2 and Stk functional domains. As shown, it can be seen that the QTL gene must be Farp2 or Stk25 (or both).

  Mice with the FARP2 821Leu allele had higher plasma HDL concentrations than mice with the FARP2 821Pro allele. To determine whether the FARP2 821Leu-Pro mutation was associated with plasma HDL level mutations, the plasma HDL concentration of 43 inbred mouse strains from the Mouse Phenome Database was analyzed. In order to avoid the strong influence of APOA2 on HDL, 43 lines were divided into 2 groups based on the important amino acid changes Ala61 to Val61 (7). In the APOA2 Ala61 group (30 lines), the HDL level of the line with allele 821Leu (12 lines) was significantly higher than that of the line with allele 821Pro in both male and female mice (18 lines) (respectively) 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 = 0.0009) (FIG. 7A). Similar trends were also found in the line with Apoa2 Vla61 (13 lines) (113.1 ± 9.8 vs 107.6 ± 9.8 mg / dL in males and 98.4 ± 11.4 pairs in females). 79.5 ± 7.9 mg / dL) (FIG. 7), but the difference did not reach statistical significance due to the small number of lines.

Example 2 Materials and Methods Animals and diet: NZW and NZB mice are obtained from The Jackson Laboratory (Bar Harbor, Maine) and mated (NZB × NZW) to produce F1 offspring, which are crossed to produce 272 F2 offspring. It was. Mice were maintained in a temperature and humidity controlled environment with a 14 hour light / 10 hour dark cycle and allowed unlimited access to food and acidified water. Freshly weaned mice are fed a standard diet (18% protein rodent diet, 6% fat, product 2018; Harlan Teklad, Madison, WI) until 8 weeks of age, then 15% milk fat, A high fat diet containing% cholesterol and 0.5% cholic acid was fed. The experimental results were reviewed and approved by the Laboratory Animal Committee of The Jackson Laboratory.

  Quantification of plasma HDL: Eight weeks after the start of the high fat diet, animals were fasted for 4 hours in the morning, bled from the retroorbital sinus in a tube containing EDTA, and centrifuged for 5 minutes at 9000 rpm. Plasma was placed in a new tube and frozen at −20 ° C. until analysis. Plasma samples were thawed within one week of blood collection, vortexed and analyzed. Plasma HDL concentration from each blood sample was measured directly using the enzyme reagent kit (# 650207, Beckman Coulter) according to the manufacturer's recommendations for the Synchron CX Delta System (Beckman Coulter).

  Genetic analysis: After proteinase K digestion, DNA was prepared from the tail sample using phenol-chloroform extraction and resuspended in 10 mM Tris.Hcl (PH: 8.0). MIT microsatellite markers for distinguishing between NZB and NZW alleles D1Mit1, D1Mit373, D1Mit212, D1Mit177, D1Mit132, D1Mit336, D1Mit218, D1Mit103, D1Mit14 and D1Mit148 (NuBios 3: Rock Gene analysis was performed on F2 progeny. SNPs rs3717961, rs3708797 and rs3022854 were genetically analyzed by Allele-Typing Service at The Jackson Laboratory in conjunction with KBiosciences (Hodsdon, UK). The location of the reported genetic map is searched from the Mouse Genome Database (see the website informatics.jax.org).

  QTL analysis: Natural log transformation of HDL values: Log transformation reduced the right asymmetry in the HDL distribution, but more importantly, it also resulted in a steady variance between hybrids. When mating populations are combined for analytical purposes, if a population has a large phenotypic variance, it tends to dominate the results. Single loci associated with HDL were detected by interval mapping. The deficient marker genotype was calculated using a multiple compensation algorithm. Unidirectional ANOVA was used to measure allelic effects at the peak QTL marker identified in the whole chromosome scan for HDL levels. A QTL is considered significant if the result meets or exceeds the 95% whole genome adjustment threshold as assessed by permutation analysis. Analysis was performed using Pseudomarker 1.1 software (see website jax.org/staff/churchill/labsite).

  Statistical combinations of hybrids: Finding repetitive QTLs in crosses with different strains suggests that they may arise from shared ancestral alleles. By recoding the B6 and NZB genotypes as a single low HDL allele and the 129 and NZW genotypes as a single high HDL allele, the raw data for Chr1 from the cross between B6 and 129 is transformed into NZB And combined with the same data from hybrids using NZW mice (Li, R. et al., Genetics, 169: 1699-1709, 2005). LOD scores were computed for each hybrid separately and then at 2-cM intervals across the QTL interval for both hybrids combined. Data were combined and analyzed with standardized “HDL phenotype” and “crossbreed” as additive covariates.

  Identification of Hdlq14 haplotypes: SNPs for use in haplotype analysis are available from SNP's extensive public databases, such as Broad SNPs (available on their website, broad.mit.edu/snp/mouse) and Perlegen SNPs (these Available on the website, mouse.perlegen.com/mouse). The NZW and 129 strains that give an allele for high HDL by comparing haplotypes from all four parental strains (NZB, NZW, B6 and 129) across a reduced interval from Mb 89.8 to 96 A genomic region that was shared between, but distinct from the NZB and B6 strains that gave an allele for low HDL was identified. The data set contained 439 SNPs ranging from 89.8 to 96 Mb, with an average spacing of SNPs of ˜14.3 kb per SNP.

  Refinement of Hdlq14 by using comparative genomics: Bioinformatic stools with all mouse and human genes aligned in an Excel sheet were found and mouse and human QTL were placed in their corresponding positions. Using this tool, the Hdlq14 region was narrowed down based on the literature.

  Microarray expression test: This reduced list of candidate genes is used to examine whether there is an expression difference between lines resulting in Hdlq14 QTL by using a microarray database. A set of microarray data from 12 lines of liver, the parent of most of the hybrids that produced 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 12 lines fed a normal or high fat diet. Search criteria for this stage indicate that the gene has significant expression differences between B6 and 129 lines (crossbred parent), but differential expression between B6 and NZB (both have the same allele for Hdlq14). It is not.

  If there is a significant sequence difference, examine the Hdlq14 positional candidates: Using the list of candidate genes expressed in the relevant tissues, the SNP database (aretha.jax.org/pub-cgi/phenome/mpdcgi? Rtn = snps / door) is searched to determine whether there are significant sequence differences. The coding region, UTR and splice site were examined. So far, 15 common inbred lines have been sequenced by Perlegen and SNPs are available at the Perlegen site and have been incorporated into other easily used sites (MGI and Ensembl). This enables a search for sequence differences in these lines in the region of interest. Sequencing was performed on three of the four lines used in the hybrids to determine Hdlq14: B6, 129 and NZW.

  Gene sequencing: Sequence differences found in the SNP database were evaluated by sequencing coding regions in all four lines 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 together at least 50 nucleotides of each exon and adjacent intron. The purified PCR product was subjected to a thermocycle sequencer and the resulting fragments were analyzed on a capillary based machine by the Jackson Laboratory DNA Sequence Laboratory. Sequence analysis was performed by aligning the sequence against the genomic B6 sequence (Sequencher version 4.1.4, GeneCodes Technology).

  QTL analysis in Chr1 from hybrids NZO × NON, B6 × C3H and Pera × DBA: Genotype and HDL data for these hybrids were from other experiments in our laboratory. Analysis was performed using Pseudomarker 1.1 software (jax.org/staff/churchill/labsite).

  Data Search: We searched for plasma HDL concentrations of 43 inbred mouse strains from the Mouse Phenome Database. Mice were fed LabDiet 5K52 (6% fat) and fasted for 4 hours before collecting their blood samples. Plasma HDL concentrations were measured with a Beckman Coulter Synchron CX5 chemical analyzer.

  Statistical difference analysis: Data were analyzed using Graphpad Prism (Windows v4.00, GraphPad Software, San Diego, CA). Plasma HDL concentrations were compared between different groups using Student's t-test.

Table 1. Positional candidates for Hdlq14

ＦＣ、倍率変化；ＦＤＲ、誤った発見割合 Table 2. Differential expression of Farp2 among B6, NZB and 129 strains fed high fat diet

FC, magnification change; FDR, false discovery rate

Table 3. Codon usage and amino acid property changes identified in Hdlq14 positional candidates

Claims (32)

  Use of an isolated antibody or a functional fragment thereof comprising an antigen binding region 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 is a cell Use to block or ameliorate the development of HDL-related diseases by binding to the above surface receptors.   The use according to claim 1, wherein the Farp gene is the Farp2 gene or a homolog or ortholog thereof.   The use according to claim 1, wherein the Stk gene is the Stk25 gene or a homolog or ortholog thereof.   A method for treating HDL-related diseases, comprising administering an effective amount of the antibody or functional fragment thereof according to any one of claims 1 to 3 to a subject.   A pharmaceutical composition comprising the antibody or functional fragment according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier or excipient suitable therefor.   A method for treating HDL-related diseases, comprising administering an effective amount of the pharmaceutical composition of claim 5 to a subject in need of treatment.   Use of an isolated antibody or functional fragment thereof for the manufacture of a medicament for the treatment of HDL-related diseases, wherein the antibody or functional fragment is a polypeptide encoded by a Farp gene or Stk gene or a homolog or ortholog thereof. Use comprising an antigen binding region that is specific for an epitope.   8. Use according to claim 7, wherein the Farp gene is Farp2 or a homolog or ortholog thereof.   The use according to claim 7, wherein the Stk gene is the Stk25 gene or a homolog or ortholog thereof.   A transgenic animal having a gene encoding the antibody or the functional fragment thereof according to claim 1.   A method of treating coronary artery disease or atherosclerosis comprising inhibiting the expression or activity of Farp or Stk or a homolog or ortholog thereof.   12. The method according to claim 11, wherein the Farp is the Farp2 gene or a homologue or orthologue thereof.   The method according to claim 11, wherein Stk is the Stk25 gene or a homolog or ortholog thereof.   The step of inhibiting Farp or Stk expression or activity further comprises an isolated antibody or functional fragment thereof comprising an antigen binding region specific for an epitope of a polypeptide encoded by the Farp or Stk gene or a homolog or ortholog thereof. 12. The method of claim 11, comprising using to inhibit activity.   15. The method of claim 14, wherein an isolated antibody or functional fragment comprising an antigen binding region binds to a surface receptor on a cell to prevent or ameliorate the development of an HDL related disease.   A method of treating coronary artery disease or atherosclerosis comprising altering the expression or activity of Farp2 or Stk25 or a homolog or ortholog thereof.   A method for detecting susceptibility to coronary artery disease or coronary artery disease, comprising detecting an allele of Farp2 or Stk25 or a homologue or ortholog thereof, which is an indicator of coronary artery disease or atherosclerosis.   18. The method of claim 17, wherein the allele is Leu821 or Pro821.   A method of measuring the efficacy of treatment of coronary artery disease or atherosclerosis, treating coronary artery disease or atherosclerosis and determining the efficacy of treatment of coronary artery disease or atherosclerosis. Or a method comprising comparing the level of Stk25 or a homolog or ortholog thereof to a control.   Method for identifying a drug useful for the treatment of coronary artery disease or atherosclerosis based on inducing an increase in plasma HDL-C levels by altering the expression or activity of FARP2 or STK25 or a homolog or ortholog thereof A biological sample is contacted with a candidate agent, and the level of total plasma lipoprotein or HDL-C in the sample is measured before and after contact with the candidate agent, the increase in HDL-C being detected in coronary artery disease. Alternatively, a method of using as an index of a drug useful for the treatment of atherosclerosis.   A method of identifying a drug useful for the treatment of coronary artery disease or atherosclerosis comprising contacting FARP2 or STK25 or a homolog or ortholog thereof with a candidate drug in the presence of a known substrate, comprising FARP2 or STK25 or A method of identifying a candidate drug as a drug useful for the treatment of coronary artery disease or atherosclerosis by altering the activity of a homolog or ortholog.   The method of claim 21, wherein the contacting step is performed on cultured cells.   24. The method of claim 21, wherein the contacting step is performed in vivo.   22. The method of claim 21, wherein Farp2 or Stk25 or a homolog or ortholog thereof is endogenous or exogenous.   A method of modulating HDL-related disease comprising administering a modulating agent that increases HDL-C levels in a subject.   26. The method of claim 25, wherein the HDL-related disease is selected from the group consisting of atherosclerosis, lipid disorders, Alzheimer's disease, oxidative stress, obesity, cardiovascular disease, type 2 diabetes and insulin resistance.   The lipid disorder is selected from the group consisting of hypercholesterolemia, dyslipidemia, hypertriglycerides, dyslipidemia, dyslipidemia, hyperlipidemia, familial hypercholesterolemia and familial hypertriglyceridemia, 27. The method of claim 26.   26. The method of claim 25, wherein the agent alters the expression or activity of FARP2 or STK25 or a homolog or ortholog thereof.   30. The method of claim 28, wherein the modulating agent is selected from the group consisting of small molecules, antisense oligonucleotides, siRNA and antibodies.   26. The method of claim 25, wherein the HDL-related disease is a disease in which the subject's HDL-C level is below commonly accepted normal HDL-C levels.   26. The method of claim 25, wherein the HDL-related disease is a disease in which the subject's HDL-C level is below the mean HDL-C level of the relevant population.   26. The method of claim 25, wherein the agent is administered with a pharmaceutically acceptable carrier.

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