WO2004094608A2 - Human procolipase like nucleic acids and polypeptides and their methods of use threof - Google Patents

Abstract

This invention relates to novel procolipase-like polynucleotides, polypeptides and antibodies. The invention also describes the related procolipase variants. Disclosed herein are the use of the novel procolipase-like polynucleotides, polypeptides and antibodies and their variants in the methods of treating overweight, obesity, dislipidemia and cardiovascular diseases. Also described herein are the uses of the polypeptide as a therapeutic with a combined effect of lipase inhibition and appetite suppression.

Description

Human Procolipase like Nucleic Acids and Polypeptides and their

Methods of Use Thereof Fitelc- of Invention

The instant invention relates to novel procolipase-like polypeptides and polynucleotides encoding the same. The invention also describes the use of procolipase as a lipase inhibitor and appetite suppressor. Thus the novel procolipase has a therapeutic utility in the treatment of obesity, diabetes, dyslipidemia and cardiovascular diseases.

BACKGROUND

Obesity is a major public health issue in the developed and developing world. It is estimated that 64% of the adult US population are either overweight or obese having a body mass index (BMI) greater than the upper limit considered normal (i.e. 25). BMI is defined as the individual's body weight in kilograms divided by the square of the height in meters. Obesity is a predisposing condition for the development of Type II diabetes, dyslipidemia and cardiovascular disease. This condition has now become rampant among school-age children as a consequence of the epidemic of obesity in that age group (See JAMA 288:1723-7, 2002). Therefore, treatments for obesity have the potential for improving glycemic control among diabetics. Reduced obese condition also resulted in lowered serum triglyceride and improved hypertension both of which are the primary risk factors for cardiovascular disease. This can be accomplished through alterations in energy metabolism and/or through a reduction in the direct toxic effects of excess circulating free fatty acids.

As compared to other dietary sources (ex: carbohydrates) dietary fat is the leading cause of overweight and obesity. Studies show that a high fat diet can produce far greater increases in body weight than a high carbohydrate diet (See Proc. Nutr. Soc. 61:299-309,

2002; Endocrine 13:167-86, 2000; Am J. Clin. Nutr. 68:1157-73, 1998; J. Nutr. 132:2488-91 , 2002). Therefore, selective reduction of dietary fat intake may benefit energy homeostasis more effectively than a reduction in caloric intake.

Current dietary interventions include diets with moderate caloric restriction, fat restriction, or very-low energy diets. Even if an initial weight loss is achieved, studies have not demonstrated conclusively that long-term weight loss can be maintained. Although increase in physical activity is not the most efficient method of initial weight loss, it is crucial for maintaining weight loss. Thus for controlling weight gain, long-term results of lifestyle modifications are disappointing because of poor compliance (See Obes. Res. Suppl 4:290S- 294S, 2001). Lipase inhibitors can drastically decrease body weight gain but could elicit unwanted side effects. Pancreatic lipase (Accession No. EC 3.1.1.3) is responsible for the digestion of dietary triacylglycerols. Several lipase inhibitors such as Orlistat (tetrahydrolipstatin), ATL- 962 (Alizyme) and teasaponin reduce weight gain by inhibiting dietary fat absorption (See Int. J. Obes. Relat. Metab. Disord., Suppl.3:S12-23, 1997; Int. J. Obes. Relat. Metab. Disord., 25:1459-64, 2001). Orlistat is the only FDA approved drug in this category. Its pharmacological activity is dose-dependent and exhibits an initial steep portion of the dose- response curve with a subsequent plateau with approximately 35% inhibition of dietary fat absorption for doses above 400 mg/day. Therapeutic doses of Orlistat (e.g. 120 mg; tid with main meals) in addition to a well balanced, hypocaloric diet, inhibit fat absorption by approximately 30% (ingested fat) resulting in an additional caloric deficit of approximately 200 calories. Specific lipase inhibitors can however cause a number of unwanted side-effects including loose faeces, oily spotting, faecal urgency and increased defecation (See Am. J. Nutr. 69:1108-16, 1999).

Recently, in vitro and in vivo studies show that the C-terminal domain (CT-domain) of porcine pancreatic lipase is a potent and specific noncovalent inhibitor of pancreatic lipase. Administration of the CT-domain reduces weight gain in rats fed a high fat diet (See J.Biol.Chem. 276:14014-8, 2001; US patent 6432400, 2002; Int. J. Obes. Relat. Metab.

Disord. 27:319-25, 2003). Rats treated with the CT-domain had significantly greater amounts of total fat (mainly triglycerides and monoglycerides) and total cholesterol in the feces than control rats. This side effect is similar to that of other lipase inhibitors. The side effects (e.g. steatorhea) resulting from inhibition of lipase activity are generally the result of unrestricted fat consumption by the individual.

Thus an effective therapeutic would be to combine an appetite suppressor and a iipase inhibitor to achieve decrease in weight gain and reduce lipase inhibition associated side effects. The current invention (referred to as CG55698 in the instant specification) provides a compound that can reduce fat intake through a central mechanism and decrease fat absorption by inhibiting pancreatic lipase activity. This will result in control of body weight gain more effectively and with reduced side effects than the known lipase inhibitors in market and in development.

Procolipase is a 12 kD polypeptide expressed in pancreas, stomach and duodenum.

Cleavage of procolipase by Trypsin results in the release of the active form of the protein (i.e. colipase) and the N-terminal pentapeptide, enterostatin. Colipase is a cofactor essential for the absorption of dietary fat. The consensus sequence of X-P-Y-P-R, where X is Ala and Y is Gly or X is Val and Y is Asp, is highly conserved across mammals. In addition, enterostatin induces serotonin and dopamine release and suppresses appetite in rats (Am. J. Physiol. 262:R1111-6, 1992; Neuroscience Letters 320:96-98, 2002; Am. J. Physiol Regul. Integr. Comp. Physiol. 278:R1346-51, 2000; Physiol. Behav. 77:5-10, 2002). Serum concentration of enterostatin is increased in response to a high fat diet but not to a high carbohydrate diet (See Obes. Res. 10:688-94, 2002). Furthermore, enterostatin is absorbed by the intestine and crosses the blood brain barrier. Peripheral or central administration of enterostatin (APGPR) selectively suppresses fat intake as compared to carbohydrate or protein in rats (See Exp. Clin. Pharmacol. 23:235-239, 2001 ; J. Gastroenterol. Suppl 14:118-27, 2002). Even though enterostatin reduces food intake in animal studies (Am. J. Physiol Regul. Integr. Comp. Physiol. 278: R1346-51 , 2000; US Patent 5494894), human clinical trials have not been successful in reducing food intake perhaps due to the inability of intravenously administered enterostatin to reach its site of action or to a sub-optimal time between administration and eating (Appetite 24:37-42, 1995). Oral administration of enterostatin to rats fed a high fat diet reduces weight gain (US Patent 5494894). This study demonstrates that oral administration of enterostatin can influence food intake and body weight. The current invention detailed in this specification describes the combination of lipase inhibitor and appetite suppressor as an effective therapeutic for obesity and obesity-related disorders such as overweight, diabetes, dyslipidemia and or cardiovascular diseases.

Summary of the Invention

It is the purpose of the invention to describe a novel procolipase deletion variant CG55698-02 or its variants as a protein therapeutic for controlling weight gain.

Another aspect of the invention is to describe a novel deletion, mutation or an insertion variants of CG55698-02 as a protein therapeutic for controlling weight gain.

In another aspect, the invention describes a polypeptide composition comprising CG55698-02 or its variants that inhibits pancreatic lipase activity as well as reduces the side effects associated with lipase inhibition in mammals.

The invention also describes a polypeptide composition comprising CG55698-02 or its variant that serves as an appetite suppressor in mammals.

Another aspect of the invention is to describe a method of inhibiting dietary fat absorption in mammals comprising administering polypeptides of CG55698-02 or its variants.

Yet another aspect of the invention is to describe a method of reducing the side effects associated with lipase inhibition by administering polypeptides of CG55698-02 or its variants. It is also the purpose of the invention to describe a method for controlling weight gain in mammals by administering a polypeptide composition of CG55698-02 or its variants that will have the combined activity of a lipase inhibitor as well as an appetite suppressor.

Another purpose of the invention is to describe a method for treating obesity, diabetes, dyslipidemia and cardiovascular diseases in mammals by administering a polypeptide composition if CG55698-02 or its variants.

The invention also encompasses chimeric protein(s) that have an immunoglobulin or a portion of the immunoglobulin tagged to the N-terminus or the C-terminus of the polypeptide of CG55698-02 or its variants.

Brief Description of the drawings

Figure 1 shows a Clustal of the pig colipase with the CG55698-02 splice variant. The arrow indicates the signal sequence cleavage site and the boxes point out the cysteines in the sequence that are bonded to each other via disulfide linkages as indicated by the lines.

Figure 2 depicts the X-ray crystal structure of the pig colipase (PDB code 1LPB;

Egloff, etal, 1995). The cysteines are shown as bonded structures in the figure (in yellow) while the missing sequence in the CG55698-02 splice variant is light blue. Figure 3 shows the amino terminus of CG55698-02 starting at residue 53 without the peptide sequence 1-11.

Figure 4 shows the pig colipase in a similar orientation to that of Figure 3, but with the deleted portions of CG55698-02 in light blue.

Figure 5 shows the x-ray crystal structure (1 ETH) at 2.84 A resolution of porcine lipase (light blue) with colipase (green) (J. Biol. Chem. 271 :18007-16, 2001). The tetra ethylene glycol monooctyl ether inhibitor (red and gray, fat tube) is shown in the active site of lipase. The deleted sequence found in CG55698-02 is highlighted in yellow.

Figure 6 shows the secondary structures of the full length colipase with the deleted sequence from CG55698-02 in yellow background. The splice variant CG55698-02 retains most of the binding sites to the C-terminal of lipase, but are missing half of the micelle interfacial binding domain and the entire N-terminal flap binding site.

Figure 7 shows the SDS-PAGE profile of limited trypsinized Wild Type CG55698-01 and CG55698-02 Colipase variant.

Figure 8 shows the specificity of an ELISA assay for human enterostatin. Figure 9 shows the effect of Limited Trypsinization of CG55698-02 and wild type

CG55698-01 on Enterostatin Release as Assessed by Human Enterostatin ELISA.

Figure 10 shows inhibitory activity of splice form (CG55698-02) and CG55698- 01 (Wild Type) protein on lipase activity Detailed Description of the invention

The present invention is based upon the discovery of novel procolipase-like polypeptides and polynucleotides encoding them. The identifier CG55698 herein generally refers to the novel procolipase-like polypeptides and polynucleotides encoding them. Table 1 describes the CG55698 and their variants.

Table 1

The CG55698 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 1 A.

Table 1A. Sequence Analysis

CG55698-02 SEQ ID NO: 1 394 bp DNA Sequence ORF Start: ATG at 17 ORF Stop: TGA at 230

AGCTGTCCCACTCGCCATGGAGAAGATCCTGATCCTCCTGCTTGTCGCCCTCTCTGTGGCCTATGCAG CTCCTGGCCCCCGGGGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAG CGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTG CCATGACGCTGGACGCTCCAAGCAGTGAGACTGCCCACCCACTCCCACACCTAGCCCAGAATGCTGTA GGCCACTAGGCGCAGGGGCATCTCTCCCCTGCTCCAGCGCATCTCCCGGGCTGGCCACCTCCTTGACC

AGCATATCTGTTTTCTGATTGCGCTCTTCACAATTAAAGGCCTCCTGCAAACCT

CG55698-02 SEQ ID NO: 2 71 aa MW at 7658.9kD Protein Sequence

MEKILIL VA SVAYAAPGPRGIIIN TLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGR SKQ CG55698-01 SEQ ID NO: 3 368 bp DNA Sequence ORF Start: ATG at 15 ORF Stop: TGA at 351

CTGTCCCACTCACCATGGAGAAGATCCTGATCCTCCTGCTTGTCGCCCTCTCTGTGGCCTATGCAGCT

CCTGGCCCCCGGGGGATCATTATCAACCTGGAGAACGGTGAGCTCTGCATGAATAGTGCCCAGTGTAA GAGCAATTGCO?GCCAGCATTCAAGTGCGCTGGGCCTGGCCCGCTGCACATCCATGGCCAGCGAGAACA GCGAGTGCTCTGTCAAGACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAGCGTGGCCTGACCTGT GAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGACG CTCCAAGCAGTOaGACTGCCCACCCACT

CG55698-01 SEQ ID NO: 4 112 aa MW at ll953.7kD Protein Sequence

MEKILI L VA SVAYAAPGPRGIIIN ENGE CMNSAQCKSNCCQHSSALGL2-RCTSMASENSECSV KTLYGIYY CPCERG TCEGD TIVGSITNTNFGICHDAGRSKQ

CG55698-03 SEQ ID NO: 5 (252 bp

DNA Sequence ORF Start: ATG at 1 |ORF Stop: TGA at 250

ATGGAGAAGATCCTGATCCTCCTGCTTGTCGCCCTCTCTGTGGCCTATGCAGCTCCTGGCCCCCGGGG GATCATTATCAACCTGTCCATGGCCAGCGAGAACAGCGAGTGCTCTGTCAAGACGCTCTATGGGATTT ACTACAAGTGTCCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACC AACACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGTGA

CG55698-03 SEQ ID NO: 6 83 aa |MW at 8912.3kD Protein Sequence

MEKILI L VALSVAYAAPGPRGIIINLSMASENSECSVKTLYGIYYKCPCERG TCEGDKTIVGSIT NTNFGICHDAGRSKQ

CG55698-05 SEQ ID NO: 7 351 bp DNA Sequence ORF Start: at 1 ORF Stop: TGA at 334

AGTGGGTGGGCAGTCTCACTGCTTGTCGCCCTCTCTGTGGCCTATGCAGCTCCTGGCCCCCGGGGGAT CATTATCAACCTGGAGAACGGTGAGCTCTGCATGAATAGTGCCCAGTGTAAGAGCAATTGCTGCCAGC ATTCAAGTGCGCTGGGCCTGGCCCGCTGCACATCCATGGCCAGCGAGAACAGCGAGTGCTCTGTCAAG ACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCAT CGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGTGAGACT GCCCACCCACT

CG55698-05 SEQ ID NO: 8 lll aa ιMW at ll700.2kD Protein Sequence

SGWAVSLLVA SVAYAAPGPRGIIINLENGELCMNSAQCKSNCCQHSSALGLARCTSMASENSECSVK iT YGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQ

CG55698-06 SEQ ID NO: 9 489 bp DNA Sequence ORF Start: ATG at 152 ORF Stop: at 401

GGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAA;

CAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGACACGAAGCTTTCTAGAAGATCT

TCGCGAGGATCCACCATGGAGAAGATCCTGATCCTCCTGCTTGTCGCCCTCTCTGTGGCCTATGCAGC

TCCTGGCCCCCGGGGGATCATTATCAACCTGTCCATGGCCAGCGAGAACAGCGAGTGCTCTGTCAAGA CGCTCTATGGGATTTACTACAAGTCACCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATC GTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGCTCGAGGG TAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCACCACCATCACCATT

GAGTTTAATTCAT

CG55698-06 SEQ ID NO: 10 83 aa MW at 8896.2kD Protein Sequence

ME I ILL VALSVAYAAPGPRGIIIN SMASENSECSV T YGIYYKSPCERGLTCEGD TIVGSIT [NTNFGICHDAGRSKQ

CG55698-07 SEQ ID NO: 11 364 bp

DNA Sequence ORF Start: ATG at 25 |ORF Stop: TAG at 274

AGAAGATCTTCGCGAGGATCCACCATGGAGAAGATCCTGATCCTCCTGCTTGTCGCCCTCTCTGTGGC

CTATGCAGCTCCTGGCCCCCGGGGGATCATTATCAACCTGTCCATGGCCAGCGAGAACAGCGAGTGCT CTGTCAAGACGCTCTATGGGATTTACTACAAGTCACCCTGTGAGCGTGGCCTGACCTGTGAGGGAGAC AAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCA GTAGCTCGAGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCACC

1ACCATCACCGGGAGTTTAATTCAT

CG55698-07 SEQ ID NO: 12 183 aa MW at 8896.2kD Protein Sequence i EKILILLLVA SVAYAAPGPRGIIINLSMASENSECSVKTLYGIYYKSPCERG TCEGDKTIVGSIT [NTNFGICHI--AGRSKQ

ICG55698-08 SEQ ID NO: 13 400 bp (DNA Sequence ORF Start: at 129 ORF Stop: at 312

AAGCTCTAGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCC

ACTGGTGACGCGGCCCAGCCGGCCAGGCGCGCGCGCCGTACGAAGCTTTCGCGAG6ATCCGGGATCAT

TATCAACCTGTCCATGGCCAGCGAGAACAGCGAGTGCTCTGTCAAGACGCTCTATGGGATTTACTACA AGTCACCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACC AACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGCTCGAGGGTAAGCCTATCCCTAACCCTCT CCTCGGTCTCGATTCTACGCGTACCGGTCATCACCACCATCACCATTGAGTTTAATTCAT

NONlh, CG55698-08 SEQ ID NO: 14 61 aa MW at 6575.4 D Protein Sequence

GIIINLSMASENSΞCSVKTLYGIYYKSPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQ

CG55698-09 SEQ ID NO: 15 1402 bp DNA Sequence ORF Start: at 122 IORF Stop: TAG at 305

AGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGi

ACGCGGCCCAGCCGGCCAGGCGCGCGCGCCGTACGAAGCTTTCGCGAGGATCCGGGATCATTATCAAC

CTGTCCATGGCCAGCGAGAACAGCGAGTGCTCTGTCAAGACGCTCTATGGGATTTACTACAAGTCACC CTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTG GCATCTGCCATGACGCTGGACGCTCCAAGCAGTAGCTCGAGGGTAAGCCTATCCCTAACCCTCTCCTC GGTCTCGATTCTACGCGTACCGGTCATCACCACCATCACCATTGAGTTTAATTCATTGATCA

CG55698-09 SEQ ID NO: 16 61 aa MW at 6575.4 D Protein Sequence

GIIIN SMASENSΞCSλ^KTLYGIYYKSPCERG TCEGDKTIVGSITNTNFGICHDAGRSKQ

CG55698-10 SEQ ID NO: 17 228 bp DNA Sequence ORF Start: ATG at 10 ORF Stop: at 223

GGATCCACCATGGAGAAGATCCTGATCCTCCTGCTTGTCGCCCTCTCTGTGGCCTATGCAGCTCCTGG

CCCCCGGGGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAGCGTGGCC TGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGAC GCTGGACGCTCCAAGCAGCTCGAG

CG55698-10 SEQ ID NO: 18 71 aa MW at 7658.9kD Protein Sequence

MEKILI LVALSVAYAAPGPRGIIINLTLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGR SKQ

CG55698-11 SEQ ID NO: 19 174 bp DNA Sequence ORF Start: at 7 ORF Stop: at 169

GGATCCGCTCCTGGCCCCCGGGGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTGTCC

CTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTG GCATCTGCCATGACGCTGGACGCTCCAAGCAGCTCGAG

1CG55698-11 SEQ ID NO: 20 54 aa MW at 5816.6kD Protein Sequence

APGPRGIIIN TLYGIYYKCPCERG TCEGDKTIVGS1TNTNFGICHDAGRSKQ

CG55698-12 SEQ ID NO: 21 |201 bp

DNA Sequence ORF Start: at 1 (ORF Stop: TGA at 199

GCTCCTGGCCCCCGGGGGATCATTATCAACCTGTCCATGGCCAGCGAGAACAGCGAGTGCTCTGTCAA GACGCTCTATGGGATTTACTACAAGTCACCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCA TCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGTGA

GCTCCTGGCCCCCGGGGGATCATTATCAACCTGTCCATGGCCAGCGAGAACAGCGAGTGCTCTGTCAA! GACGCTCTATGGGATTTACTACAAGTCACCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCA TCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGAGATCT --AT_GCT_CCAG_GT_GGTCAT_CATCAT_CA_TCATCAT_TAFI_,

CG55698-13 SEQ ID NO: 24 79 aa MW a 8516.4kD Protein Sequence

APGPRGIIINLSMASENSECSVKTLYGIYYKSPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQRS (NAPGGHHHHHH

CG55698-14 SEQ ID NO: 25 189 bp DNA Sequence ORF Start: at 1 ORF Stop: TAA at 187

GGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAGCGTGGCCTGACCTG TGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGAC GCTCCAAGCAGAGATCTAATGCTCCAGGTGGTCATCATCATCATCATCATTAA

CG55698-14 SEQ ID NO: 26 62 aa MW at 6800.6kD Protein Sequence

GIIINLTLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQRSNAPGGHHHHHHi

CG55698-18 SEQ ID NO: 27 180 bp DNA Sequence ORF Start: ATG at 7 ORF Stop: end of sequence

CATACCATGGCTCCTGGCCCCCGGGGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTG TCCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACT TTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGCTCGAGGGC

CG55698-18 SEQ ID NO: 28 58 aa MW at 6247.1kD Protein Sequence

IMAPGPRGIIINLTLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQ EG

CG55698-20 SEQ ID NO: 29 225 bp DNA Sequence ORF Start: at 1 ORF Stop: end of sequence

AAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCGCTCC TGGCCCCCGGGGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAGCGTG GCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCAT GACGCTGGACGCTCCAAGCAG

CG55698-20 SEQ ID NO: 30 75 aa MW at 7896.1kD Protein Sequence

KYLLPT-i-AAGLLLLAAQPAMAAPGPRGIIIN TLYGIYYKCPCERG TCEGDKTIVGSITNTNFGICH DAGRSKQ CG55698-01 (SEQ ID NO: 4) is the human colipase precursor identified as ACC:P04118

SWISSNEW protein data base. The novel deletion variant CG55698-02 (SEQ ID NO: 2) in its mature form is described herein as CG55698-11 (SEQ ID NO: 20). Further the mature form can be generated with tandem repeats of enterostatin (APGPR) to enhance the functionality and may be tagged with linker proteins, Histidine repeats (His tags) portions of immunoglobulin (Ex: Fc, V5) thioredoxin protein, maltose binding protein, FLAG or MSA in the N-terminus or the C-terminus. The use of tags to facilitate secretion, purification or enhance the stability of the protein is well known in the art and can be readily identified by one skilled in the art. Examples of the variants are herein described:

1) Nucleotide sequence of CG55698-22 GCCCCTGGTCCACGTGCACCAGGCCCTCGCGCTCCTGGTCCACGTGGGATCATTATCAACCTGACGCTCT ATGGGATTTACTACAAGTGTCCC-.GTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTC CATCACCA^CACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGGACTACAAGGACGATGAC GACAAGTAATAAGTCGACGGC (SEQ ID NO: 31) Protein sequence of CG55698-22

APGPRAPGPRAPGPRGIIINLTLyGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQDYKDDD

DK (SEQ ID NO: 32)

SEQ ID NO: 31 and 32 described above show three tandem repeats of enterostatin at the N- terminus and tagged to FLAG protein with the sequence DYKDDDDK at the C-terminus.

2) Nucleotide sequence of CG55698-23

GCCCCTGGTCCACGTGCACCAGGCCCTCGCGCTCCTGGTCCACGTGCACCAGGCCCTCGCGCCCCTGGTC CACGTGGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAGCGTGGCCTGAC CTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGA CGCTCCAAGCAGGACTACAAGGACGATGACGACAAGTAATAAGTCGACGGC (SEQ ID NO: 33)

Protein sequence of CG55698-23

APGPRAPGPRAPGPRAPGPRAPGPRGIIINLTLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAG

RSKQDYKDDDDK (SEQ ID NO: 34) SEQ ID NO: 33 and SEQ ID NO: 34 described above show five tandem repeats of enterostatin at the N-terminus and tagged to FLAG protein with the sequence DYKDDDDK at the C-terminus.

3) Nucleotide sequence of CG55698-24 GCCCCTGGTCCACGTGCACCAGGCCCTCGCGCTCCTGGTCCACGTGGGATCATTATCAACCTGACGCTCT ATGGGATTTACTACAAGTGTCCCTGTGAGCGTGGCCTGACCTGTGAGGGAGACAAGACCATCGTGGGCTC CATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGACGCTCCAAGCAGCACCACCACCACCACCAC

TAGCTCGAGGGC (SEQ ID NO: 35)

Protein sequence of CG55698-24

APGPRAPGPRAPGPRGIIINLTLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQHHHHHH (SEQ

ID NO: 36)

SEQ ID NO: 35 and SEQ ID NO: 36 described above show three tandem repeats of enterostatin at the N-terminus and is tagged to 6-Histidines at the C-terminus.

4) Nucleotide sequence of CG55698-25

GCCCCTGGTCCACGTGCACCAGGCCCTCGCGCTCCTGGTCCACGTGCACCAGGCCCTCGCGCCCCTGGTC CACGTGGGATCATTATCAACCTGACGCTCTATGGGATTTACTACAAGTGTCCCTGTGAGCGTGGCCTGAC CTGTGAGGGAGACAAGACCATCGTGGGCTCCATCACCAACACCAACTTTGGCATCTGCCATGACGCTGGA CGCTCCAAGCAGCACCACCACCACCACCACTAGCTCGAGGGC (SEQ ID NO: 37)

Protein sequence of CG55698-25 APGPRAPGPRAPGPRAPGPRAPGPRGIII TLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAG RSKQHHHHHH (SEQ ID NO: 38)

SEQ ID NO: 37 and SEQ ID NO: 38 described above show five tandem repeats of enterostatin at the N-terminus and is tagged to 6-Histidines at the C-terminus.

Further analysis of the CG55698-02 protein yielded the following properties shown in Table 1 B.

Table IB. Protein Sequence Properties CG5569§-02

SignalP analysis: Cleavage site between residues 18 and 19

PSORT H analysis:

PSG: a new signal peptide prediction method

N-region: length 3; pos.chg 1; neg.chg 1 H-region: length 18; peak value 11.30 PSG score: 6.90

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 7.41 possible cleavage site: between 17 and 18

>» Seems to have a cleavable signal peptide (1 to 17)

ALOM: Klein et al's method for TM region allocation Init position for calculation: 18

Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 5.67 (at 23) ALOM score: 5.67 (number of TMSs: 0)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 8 Charge difference: 1.0 C( 2.0) - N( 1.0) C > N: C-terminal side will be inside

>»Caution: Inconsistent mtop result with signal peptide MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 7.92 Hyd Moment (95): 8.90 G content: 3 D/E content: 2 S/T content: 2 Score: -6.38

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 32 PRG|lI

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 9.9% NLS Score: -0.47

KDEL: ER retention motif in the C-terminus: none

ER Membrane Retention Signals:

KKXX-like motif in the C-terminus: GRSK S L: peroxisomal targeting signal in the C-terminus: none

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

Actinin-type actin-binding motif: type 1 : none type 2 : none

NMYR: N-myristoylation pattern : none

Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none

Tyrosines in the tail: none

Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 89

COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues

Final Results (k = 9/231

44.4 %: endoplasmic reticulum

33.3 %: extracellular, including cell wall

11.1 % : vacuolar

11.1 % : mitochondrial

» prediction for CG55698-02 is end (k=9)

A search of the CG55698-02 protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table ic

Table IC. Geneseq Results for CG55698-02

Identities/

CG55698-02 Similarities

Geneseq Protein/Organism/Length Residues/ Expect for the Identifier [Patent #, Date] Match Value Matched Residues Region

In a BLAST search of public sequence databases, the CG55698-02 protein was found to have homology to the proteins shown in the BLASTP data in Table 1 D.

PFam analysis predicts that the NOV1 a protein contains the domains shown in the Table 1 E. Table IE. Domain Analysis of CG55698-02

Identities/

Pfam CG55698-02 Match Similarities Expect Domain Region for the Matched Value

Region

Colipase C 21..65 34/47 (72%) 3.1e-24 37/47 (79%)

As would be understood by those skilled in the art, a nucleic acid or amino acid sequence homologous to the CG55698 sequences (or its variants) disclosed herein could be used in the method of the invention. "Homologous" refers to sequences characterized by a homology at the nucleotide level or amino acid level and include sequences coding for isoforms such as those expressed in different tissues of the same origin encoded for example by alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous sequences include CG55698 polypeptides and the nucleotide sequences encoding them, of species other than humans, including, but not limited to vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat, cow, horse, and other organisms. Homologous sequences also include naturally occurring allelic variations and mutations of the sequences set forth herein. Homologous sequences include those sequences having conservative amino acid substitutions and the nucleotides encoding them. A polypeptide having a biologically active portion of a CG55698 polypeptide has an activity of CG55698 as measured in a particular biological assay (such as those described herein). Such biologically active polypeptides may be used in the method of the invention. A nucleic acid fragment encoding a biologically-active polypeptide can be prepared by isolating a portion of the nucleotide that encodes a polypeptide having a CG55698 biological activity, expressing the encoded portion of CG55698 (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of CG55698.

A polypeptide agent is meant to include, e.g., any substance that can prevent binding of CG55698 wild type variant to pancreatic lipase and suppress appetite. The agent of this invention preferably can change an aspect of fat metabolism and appetite. Such change can be the result of any of a variety of events, including, e.g., preventing or reducing interaction between CG55698 wild type variant and pancreatic lipase, central effects on macronutrient choice.

Non-sequence modifications include, e.g., in vivo or in vitro chemical derivatizations of CG55698 variant. Non-sequence modifications include, e.g., changes in phosphorylation, acetylation, methylation, carboxylation, or glycosylation. Methods for making such modifications are known to those skilled in the art. For example, phosphorylation can be modified by exposing CG55698 variant to phosphorylation-altering enzymes, e.g., kinases or phosphatases. Preferred analogs include CG55698 variant or biologically active fragments thereof whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish CG55698 variant biological activity. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

The protein compositions can further be analogs of the polypeptides described. Analogs are meant to include peptides in which structural modifications have been introduced into the peptide backbone so as to make the peptide non-hydrolyzable. Such peptides are particularly useful for oral administration, as they are not digested. Peptide backbone modifications include, e.g., modifications of the amide nitrogen, the α-carbon, the amide carbonyl, or the amide bond, and modifications involving extensions, deletions or backbone crosslinks. For example, the backbone can be modified by substitution of a sulfoxide for the carbonyl, by reversing the peptide bond, or by substituting a methylene for the carbonyl group. Such modifications can be made by standard procedures known to those skilled in the art. See, e.g., Spatola, A. F., "Peptide Backbone Modifications: A Structure- Activity Analysis of Peptides Containing Amide Bond Surrogates, Conformational Constraints, and Related Backbone Replacements," in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein (ed.), Marcel Dekker, Inc., New York (1983). Analogs also include polypeptides in which one or more of the amino acid residues include a substituent group, or polypeptides which are fused with another compound, e.g., a compound to increase the half-life of the polypeptide, e.g., polyethylene glycol.

The polypeptide compositions can further encompass fragments of the polypeptides described in this applicaton. By fragment it is meant some portion of the CG55698 variant polypeptide. Fragments include, e.g., truncated secreted forms, proteolytic fragments, splicing fragments, other fragments, and chimeric constructs between at least a portion of the relevant gene, e.g., CG55698 variant, and another molecule. Fragments of CG55698 variant can be generated by methods known to those skilled in the art. In certain embodiments, the fragment is biologically active. The ability of a candidate fragment to exhibit a biological activity of CG55698 variant can be assessed by methods known to those skilled in the art. For example, CG55698 variant fragments can be tested for their ability to inhibit pancreatic lipase, as described herein. Also included are CG55698 variant fragments containing residues that are not required for biological activity of the fragment or that result from alternative mRNA splicing or alternative protein processing events. Fragments of a protein can be produced by any of a variety of methods known to those skilled in the art, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNAs which encode an array of fragments. DNAs which encode fragments of a protein can also be generated; e.g., by random shearing, restriction digestion or a combination of the above- discussed methods. For example, fragments of CG55698 variant can be made by expressing CG55698 variant which has been manipulated in vitro to encode the desired fragment, e.g., by restriction digestion of any of the DNA sequences described herein in the instant application. Fragments can also be chemically synthesized using techniques known in the art, e.g., conventional Merrifield solid phase f-Moc ort-Boc chemistry for example, peptides of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

An CG55698 variant or a biologically active fragment or analog thereof, or a binding molecule or a biologically active fragment or analog thereof, can, e.g., compete with its cognate molecule for the binding site on the complementary molecule, and thereby reduce or eliminate binding between CG55698 wild type variant and pancreatic lipase. CG55698 variant or binding molecule can be obtained, e.g., from purification or secretion of naturally occurring CG55698 variant or binding molecule, from recombinant CG55698 variant or binding molecule, or from synthesized CG55698 variant or binding molecule. Methods for generating analogs and fragments and testing them for activity are readily available and known to those skilled in the art.

A composition described herein can be an agent such as a nucleic acid used as an antisense molecule. Antisense therapy is meant to include, e.g., administration or in situ generation of oligonucleotides or their derivatives which specifically hybridize, e.g., bind, under cellular conditions, with the cellular mRNA and/or genomic DNA encoding an CG55698 variant polypeptide, or mutant thereof, so as to inhibit expression of the encoded protein, e.g., by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.

In certain embodiments, the antisense construct binds to a naturally-occurring sequence of an CG55698 variant gene which, e.g., is involved in expression of the gene. These sequences include, e.g., promoter, start codons, stop codons, and RNA polymerase binding sites. In other embodiments, the antisense construct binds to a nucleotide sequence which is not present in the wild type gene. For example, the antisense construct can bind to a region of an CG55698 variant gene which contains an insertion of an exogenous, non-wild type sequence. Alternatively, the antisense construct can bind to a region of an CG55698 variant gene which has undergone a deletion, thereby bringing two regions of the gene together which are not normally positioned together and which, together, create a non-wild type sequence. An antisense construct of the present invention can be delivered, e.g., as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an CG55698 variant polypeptide. An alternative is that the antisense construct is an oli gonucleotide which is generated ex vivo and which, when introduced into the cell causes in i bition of expression by hybridizing with the mRNA (duplexing) and/or genomic sequences (tri plexing) of a CG55698 variant gene. Such oligonucleotides are preferably modified oli gonucleolides which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate, phosphorodithioates and methylphosphonate analogs of DNA and peptide nucleic acids (PNA). (See also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed. (See, e.g., Van der Krol et al., Biotechniques 6: 958-976, (1988); Stein et al., Cancer Res. 48: 2659-2668 (1988)). Compositions also include inhibitors of a molecule that are required for synthesis, post-translational modification, or functioning of CG55698 variant and/or a binding molecule, or activators of a molecule that inhibits the synthesis or functioning of pancreatic lipase. Agents include, e.g., cytokines, chemokines, growth factors, hormones, signaling components, kinases, phosphatases, homeobox proteins, transcription factors, editing factors, translation factors and post-translation factors or enzymes. Agents are also meant to include ionizing radiation, non-ionizing radiation, ultrasound and toxic agents which can, e.g., at least partially inactivate CG55698 wild type variant and/or pancreatic lipase. An agent is also meant to include an agent which is not entirely CG55698 variant specific. For example, an agent may alter other genes or proteins related to intestinal fat absorption. Such overlapping specificity may provide additional therapeutic advantage.

In addition to naturally-occurring allelic variants of CG55698 sequences, the skilled artisan will further appreciate that changes may be introduced into the nucleotide sequences that may lead to changes in the amino acid sequences of the encoded CG55698 protein, without altering the functional ability of that protein (Ex: CG55698-03, enterostatin tandem repeats, CG55698-22, CG55698-23, CG55698-24, CG55698-25 as described herein). Such proteins also have utility in the method of the invention. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the protein without altering its biological activity, whereas an "essential" amino acid residue is required for such biological activity. Amino acids for which conservative substitutions can be made are well-known within the art. Polypeptides that contain changes in amino acid residues of CG55698 polypeptides that are not essential for activity may also be used in the method of the invention. Furthermore, structure-based modifications that enhance the functional activity may result in the amino acid sequence alteration of CG55698 (Ex: CG55698-03, as described herein).

Chimeric or fusion proteins including CG55698 polypeptides may be used in the method of the invention. As used herein, a "chimeric protein" or "fusion protein" comprises a CG55698 polypeptide operatively-linked to a non-CG55698 polypeptide. Within such a fusion protein the CG55698 polypeptide can correspond to all or a portion of a CG55698 protein. In one embodiment, a CG55698 fusion protein comprises at least one biologically-active portion of a CG55698 protein. Within the fusion protein, the CG55698 polypeptide and the non-CG55698 polypeptide are "operatively-linked", that is they are fused in-frame with one another. The non-CG55698 polypeptide can be fused to the N-terminus (Ex: CG55698-01 His, mature CG55698-02 His) or C-terminus (Ex: CG55698-01 His, mature CG55698-02 His, CG55698-24, CG55698-25) of the CG55698 polypeptide. For example, the fusion protein may be a CG55698 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of CG55698 can be increased through use of a heterologous signal sequence. In yet another example, the fusion protein is a CG55698-immunoglobulin fusion protein in which the CG55698 sequences are fused to sequences derived from a member of the immunoglobulin protein family. The CG55698-immunoglobulin fusion proteins can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an immunological response according to the present invention. A CG55698 chimeric or fusion protein for use in the method of the invention may be chemically modified for the purpose of improving bioavai lability, and increasing efficacy, solubility and stability. For example, the protein may be covalently or non-covalently linked to polyethylene glycol (PEG), or by other well-known methods in the art.

A CG55698 chimeric or fusion protein for use in the method of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences may be ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. Furthermore, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence [see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, (1992)]. Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A CG55698-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CG55698 protein. Furthermore, the chimeric or fusion protein may be produced synthetically. The fusion protein can be a CG55698 protein fused to a His tag (Ex: CG55698-01 , CG55698-02, CG55698-06, CG55698-10, CG55698-13, CG55698-14, CG55698-24, CG55698-25), epitope tag (e.g. V5, CG55698-02, CG55698-10) or Fc tag to aid in the purification and detection of the recombinant CG55698 protein. The coding sequence can also be ligated into expression vectors such as yeast (Ex: CG55698-01, CG55698-02, CG55698-10), E.coli (Ex: CG55698-06) mammalian cells (Ex: CG55698-02, CG55698-10), insect cells or any other expression vector systems that are known to those skilled in the art.

In one use, the present invention provides CG55698 proteins, analogs and homologs that can be incorporated into pharmaceutical compositions suitable for administration for patient use. Such compositions comprise the CG55698 polypeptide according to the present invention, either alone or together with one or more conventional pharmaceutically acceptable carriers, such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound which is the CG55698 protein as described herein, use thereof in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

In another embodiment, the method uses a pharmaceutical composition formulated to be compatible with its intended route of administration. Examples of routes of administration include but not limited to oral or parenteral i.e intravenous, intranasal, transmucosal or transdermal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

By intranasal administration, it is meant that the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Pharmaceutical compositions suitable for injectabie use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectabie solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition

By carriers, as used herein it is meant that the composition is prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations are readily apparent to those skilled in the art. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods well-known to those skilled in the art.

Antibodies are meant to include CG55698 variant antibodies against any moiety that directly or indirectly affects pancreatic lipase activity. The antibodies can be directed against, e.g., CG55698 wild type variant pancreatic lipase binding epitope, or a subunit or fragment thereof. For example, antibodies include CG55698 variant antibodies; and anti-binding molecule antibodies. Antibody fragments are meant to include, e.g., Fab fragments, Fab' fragments, F(ab').sub.2 fragments, F(v) fragments, heavy chain monomers, heavy chain dinners, heavy chain trimers, light chain monomers, light chain dimers, light chain trimers, dimers consisting of one heavy and one light chain, and peptides that mimic the activity of the CG55698 variant or anti-binding molecule antibodies. For example, Fab2 ' fragments of the inhibitory antibody can be generated through, e.g., enzymatic cleavage. Both polyclonal and monoclonal antibodies can be used in this invention. Preferably, monoclonal antibodies are used. Natural antibodies, recombinant antibodies or chimeric-antibodies, e.g., humanized antibodies, are included in this invention. Preferably, humanized antibodies are used when the subject is a human. Most preferably, the antibodies have a constant region derived from a human antibody and a variable region derived from an inhibitory mouse monoclonal antibody. Production of polyclonal antibodies to CG55698 variant can be performed by standard procedures known in the art. Monoclonal and humanized antibodies are generated by standard methods known to those skilled in the art. Monoclonal antibodies can be produced, e.g., by any technique which provides antibodies produced by continuous cell lines cultures. Examples include the hybridoma technique (Kohler and Milstein, Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, A. R. Lisa, Inc., pp. 77- 96 (1985)). Preferably, humanized antibodies are raised through conventional production and harvesting techniques (Berkower, I., Curr. Opin. Biotechnol. 7:622-628 (1996); Ramharayan and Skaletsky, Am. Biotechnol. Lab 13:26-28 (1995)). In certain preferred embodiments, the antibodies are raised against the CG55698 wild-type variant, preferably the pancreatic lipase binding site, and the Fab fragments produced. These antibodies, or fragments derived therefrom, can be used, e.g., to block pancreatic lipase activity by inhibiting CG55698 wild- type variant.

The data included herein support the use of the nucleic acids, polypeptides and antibodies in the treatment of overweight, obesity, diabetes, dyslipidemia and other cardiovascular diseases as described above. Briefly the present invention provides a novel procolipase-like polypeptides and polynucleotides encoding them that exhibit lipase inhibition and appetite suppression associated with weight gain in mammals, and use of the same. Furthermore, the present invention describes compositions comprising a therapeutic polypeptide, CG55698, useful in controlling weight gain. More specifically, the novel compositions detailed in the present invention are useful in obesity-related disorders as detailed in the instant specification.

Example 1. Three Dimensional Analysis of the Novel Deletion Variant CG55698-02

A procolipase-like gene was identified and designated CG55698-02, (SEQ ID NO: 1 , SEQ ID NO: 2, USSN: 60/323519 and USSN: 10/236392; WO200270539). This novel, 71- amino acid polypeptide, is a splice variant that lacks amino acids 29 to 69 present in the wild type procolipase. This splice variant represents a novel functional colipase precursor-like protein.

The three dimensional structure of the deletion variant was compared with the pig procolipase (Clustal provided in Figure 1) using the Washington University Blast Archives (UWBIast2.0) (Cn3D 4.1; Swiss PDB Viewer).

Based on the predicted three-dimensional structure of CG55698-02 (Figure 2- Figure 5), it is likely that the N-terminal pentapeptide APGPR and the remaining deletion variant of colipase can be generated by cleavage with intestinal Trypsin. The truncated colipase is likely to interact with lipase; however, it lacks the lipase activation function found in the wild type (full length colipase). In the duodenum, the interaction between CG55698-02 and pancreatic lipase may prevent the interaction between full-length wild type colipase and the pancreatic lipase and, hence, acts as a competitive inhibitor of total pancreatic lipase activity thereby reducing fat absorption in the intestine.

From Figure 2, it can be concluded that cysteines 17, 23, 27, 28, 39, and 49 (relative to the mature colipase) are deleted in CG55698-02. Cys27 and Cys49 are involved in disulfide linkages with the non-deleted part of the protein. Thus, CG55698-02 would leave two unpaired cysteine residues (Cys61 and Cys69). Figure 2 shows a basic model of GG55698- 02 without residues 1-11 on the amino terminus. The deletion of the sequences in CG55698- 02 would result in only one disulfide bond between Cys63 and Cys87 to hold the protein together. The lack of other disulfide bonds suggests that the resulting CG55698-02 would be more flexible than the mature colipase. Although Cys 61 and Cys 69 are not paired in the mature colipase, it is conceivable that with an adjustment of the phi and psi torsion angles of the carbon backbone between Cys61 and Cys63 (light blue circular arrow), that Cys61 could be rotated to the appropriate 2 A distance required to form a disulfide bond with Cys69. Such a conformational alteration might pair up the free cysteines in a disulfide bond and thus add stability to the CG55698-02 structure.

Figure 3 also shows the amino terminus of CG55698-02 starting at residue 53 without the peptide sequence 1-11. Figure 4 shows the pig colipase in a similar orientation to that of Figure 3, but with the deleted portions of CG55698-02 in light blue. The colipase sequence proximal to the amino terminus shown in Figure 4 forms an antiparallel β sheet that is secured in place by a disulfide bond between Cys49 and Cys69.

Since the peptide sequence 1-11 in CG55698-02 lacks a cysteine, it cannot form a disulfide bond to secure the peptide in place. It is not clear what the conformation of residues 1-11 would be in the CG55698-02 structure which is missing -43% of the protein sequence relative to the mature colipase. This suggests that for CG55698-02, it would be very unlikely that this peptide sequence would be buried in a nonsolvent accessible conformation.

Also, this sequence appears to be very flexible in the mature colipase, since it does not adopt a regular secondary structure and appears to be very solvent exposed (Figure 2). All of this data would suggest that peptide sequence 1-11 in CG55698-02 is still highly flexible, highly solvent exposed and therefore labile to proteolytic digestion. The deletion variant, therefore, is likely to be acid resistant and accessible for proteolytic digestion.

CG556Θ8-02 May Retain the Binding Capability to Pancreatic Lipase and serve as a

Competitive Inhibitor

Pancreatic lipase catalyzes the hydrolysis of triacylglycerol to fatty acids in the intestine. These triacylglycerides are present predominantly as an emulsified micelle stabilized by bile acids. In order for pancreatic lipase to hydrolyze the ester linkage of triacylglycerides, the active site must be positioned at the bile salt-coated water-lipid interface of the micelle. Bile salts inhibit pancreatic lipase binding of colipase to the micelle anchors the lipase to the water-lipid interface so that hydrolysis can occur.

Homology between porcine and human lipase is very high. Therefore, it is feasible to use the x-ray crystal structure of the porcine lipase to model the effects of CG55698-02 on lipase binding. Figure 5 shows the x-ray crystal structure (1ETH) at 2.84 A resolution of porcine lipase (light blue) with colipase (green) (See J. Biol. Chem. 271:18007-16, 2001). The tetra ethylene glycol monooctyl ether inhibitor (red and gray, fat tube) is shown in the active site of lipase. The deleted sequence found in CG55698-02 is highlighted in yellow. The amino-terminal domain of lipase contains the active site whereas the carboxy- terminal domain binds to colipase. Likewise, colipase possesses a lipase binding domain and a micelle interfacial binding site. The catalytic site of lipase is inaccessible in solution since there is an N-terminal flap that covers the active site, preventing substrate from entering. Additionally, the colipase serves to stabilize the active form of lipase by binding to the N- terminal flap and thus keeping it in an open, active conformation enabling the substrate to enter the lipase catalytic site.

The interfacial binding site of colipase is composed of four hydrophobic fingers (finger 1:14- 24, finger 2: 27-39, finger 3: 47-64, and finger 4: 68-90 numbered according to the colipase sequence in Figure 1). In CG55698-02, Fingers 1 , 2 and a portion of 3 are missing suggesting that the splice variant would be less adept at binding the micelle interface.

Of the eight polar interactions (that include hydrogen bonds and salt bridges) between lipase and colipase, five bind to the C-terminal region of lipase and the remainder bind to the N-terminal flap. Of these, only one of the 5 bonds:C-terminal bonds in CG55698- 02 is missing, but all three of the:N-teriminal flap bonds in CG55698-02 are missing. Of the 17 colipase:lipase van der Waals contacts, 4 of these contact the N-terminal flap and the remainder bond to the C-terminal domain. For CG55698-02, 11 of the 13 van der Waals contacts to the lipase C-terminal domain and none of the N-terminal flap contacts are present. Of the 4 bridging water contacts at the colipase: lipase C-terminal binding site, 2 are lost in CG55698-02 as shown in Figure 6

Even though, the splice variant CG55698-02 retains most of the binding sites to the C-terminal of lipase, is missing half of the micelle interfacial binding domain and the entire N- terminal flap binding site, it could still bind to pancreatic lipase. Furthermore, it may not anchor pancreatic lipase to the micelle interface very well and would not be able to stabilize the open, active conformation of pancreatic lipase (since it cannot bind the N-terminal flap). Therefore, it is possible that CG55698-02 could compete for binding with the wild-type, lipase- activating form, of colipase. Since binding of CG55698-02 to pancreatic lipase may fail to position the N-terminal flap away from the active site of lipase, preventing substrate binding, CG55698-02 could function as a natural competitive inhibitor of pancreatic lipase.

Furthermore addition of the missing amino acids could potentially increase the binding capacity of CG55698-02. Thus based on the predicted lipase-colipase complex structure detailed above, CG55698-03 variant was created. Comparison of CG55698-01 , human procolipase (wild-type) (CG5569801 , SEQ ID NO:4) (SWISSPROT-ACC:P04118), CG55698-02, the splice variant of the current invention (CG5569802, SEQ ID NO:2) and CG55698-03 (CG5569803, SEQ ID NO:6) is shown in Table 2

Table 2: CLUSTAL of Procolipase and their Variants

1. CG55698-01-PROT

2. CG55698 02-PROT

3. CG55698-03-PROT cg5--ό9802ρrot ΛEKILI LLLVALSVAYAAPGFRGI I INL cg5->69803ρrot jj ΛEKILILLLVALSVAYAAPGFRGI I IN cg5569801ρrot |] ΛEKILIL LVALSVAYAAPGPRGI I INI lENGELCMNSAQCKSNCCQHSSALGLARCTg

Example 2. Expression Vector for the Production of Recombinant CG55698-02 Protein Recombinant CG55698-02 protein was expressed using the in-frame to the

CG55698-02 nucleotide sequence. The coding sequence was inserted into various expression vectors such that the encoded proteins can be produced. The expressed proteins were either full-length CG55698-02 protein or any derivative of CG55698-02 that includes more amino acids in the colipase sequence that are non-naturally occurring to increase its binding activity to lipase based on the predicted lipase-colipase complex structure (See

Biochim. Biophys. Acta, 1441 :173-84, 1999). This may include but not limited to one or more constructs described in the instant application. The recombinant protein was purified by published protocol (See Protein Expr. Purif. 5:583-6, 1994) or combination of techniques readily available to the skilled person in the art. A His-tag was attached to the C-terminus (Ex: CG55698-02 C terminal tag) or the N-terminus (Ex: CG55698-02 N terminal tag) of the resultant recombinant protein to enable metal affinity purification. Table 3. Schematic representation of the constructs generated

CG55698- Novel Yeast, 3648 02 (exon 2 deleted)

CG55698" CHO, 3870 10

CG55698- Partial deletion; 06 C49S mutation

E.coli, 3857 p I Native secreted signal, Yeast c factor, enterostatin, | | colipase, H C49, || S49, ^§ His tag, ■I V5 tag

While Table 3 illustrates some of the constructs generated, other constructs with further modification of the CG55698 variants (Ex: with tandem repeats of enterostatin, CG55698-02 variants 1-4, SEQ ID NO: 31-34) could be generated as detailed in the specification.

Example 3. Confirmation of the Accessibility for Trypsin Digestion

The purified protein CG55698-02 or its derivative was subjected to trypsin digestion in vitro according to the published protocol or modifications thereof (Biochim.Biophys. Acta 671 : 155-63, 1981). The resulting peptides were analyzed using either a reverse phase HPLC or mass spectroscopy method to demonstrate that a pentapeptide corresponding to the N- terminal of the CG55698-02 was cleaved and a truncated colipase or mature CG55698-02 was generated.

Trypsinization Protocol

This procedure was used to digest CG55698 variants (Ex. CG55698-02) in order to generate protein segments or peptides from said protein exhibiting specific biological activities.

Digestion was carried out as follows: Immobilized TPCK-trypsin (Pierce Biotechnology; Rockport, II) was washed 10x (100 μl of 50% resin slurry to 1 ,400 μl buffer; 400xg; 10 min; 4°C; each wash) with digestion buffer (0.05 M Ammonium Bicarbonate, pH 8.5). Digestion conditions were optimized (e.g. trypsin concentration; duration of digestion). Reaction components consisted of CG55698 variants (in 20 mM Tris/150 mM NaCI), washed/immobilized TPCK-trypsin, and digestion buffer (20 mM Tris/150 mM NaCI). Trypsinization efficiency was assessed via SDS-PAGE and/or HPLC-MALDI and or Enterostatin ELISA. SDS-PAGE analysis of the digest showed that CG55698-02 with a 6 His C-terminal tag was more resistant to trypsinization that the wild type form (CG55698-01) (Figure 7). Addition of a 6 His Tag to the N-terminus of CG55698-02 resulted in a form of CG55698-02 highly resistant to trypsinization. Although C-terminal tag CG55698-02 was more resistant to trypsinization than the wild-type (CG55698-01), the trypsin cleavage site at the N-terminal of the splice variant was still accessible as evidence by the presence of Enterostatin peptide in the digest (see Enterostatin ELISA, Figures 8 and 9; and HPLC- MALDI, Table 4).

HPLC-MALDI Samples were resuspended in 100 μl of 0.1% TFA. A 10 μl aliquot of each trypsinized sample and each control sample was used for Zip-tip sample clean up prior to samples elution and MALDI target plate spotting. For sample clean-up C18 reverse-phase Zip-tips were used. Generally, C18 Zip-tips are applicable when used for low molecular weight proteins and peptides (less than 50,000 Da). C18 tips were washed before sample binding in the following order: once with 10 μl acetonitrile; once with 10 μl 1:1 (Acetonitrile : 0.1% TFA); twice with 10 μl 0.1%TFA. Samples were bound to the C18 Zip-tip by gentle pipetting of the sample into the reverse phased tips 7 times. Once sample was bound to the Zip-tip, the tips were further washed three times with 0.1%TFA. Samples were then eiuted into 4 μl of 10mg/ml CHCA matrix (1:1 Acetonitrile/0.1%TFA), and two duplicate 2 μl spots were placed onto the MALDItarget plates. Dried samples were run in reflectron mode on MALDI-TOF-MS (Micromass Waters MALDI-TOF-LR (LR-linear and reflectron detectors) Milford, MA). Data files were loaded into Mascot using the Protein 3 database for Trypsin samples with a maximum of 3 missed cleavages. Fixed modifications were set to carboxymethyl (C) and Oxidation (M). Peptide tolerance was set to +/- 1 Da based on MH+ monoisotopic values while the protein mass was left unrestricted. Reports were set to search for the top 20 hits. Trypsinized C-terminal tag CG55698-02 is more resistant to trypsinization than the wild-type (CG55698-01). Furthermore, the trypsin cleavage site at the N-terminal of the C-terminal tag splice variant was still accessible as evidence by the presence of Enterostatin peptide in the digest as shown by the arrow in Table 4.

Table 4. Confirmation of Enterostatin Release from Limited Trypsinization of CG55698- 02 wa ALDI

Fixed --odi--ica_ion-s : Carba-nidor-ethyl (C)

Variable modifica ons: Oxidation (H)

Cleavage by Trypsin: cuts C-term side of KR unless next residue is P

Nu-Rber of mass values searched- 74-

Number of mass values Matched: 12

Sequence Coverage: 100%

Hatched peptides shora in Bn- l Bert

1 ---B1RSIIIB LI XBISSKC PCERGI-TCEt. -.KTIW-SISSr ---FβZCHD-U. 51 RSRQRSESPe εHJMHHH

Show predicted peptides also

SortPeptides By | <* Residue Number C lαcreasit--. Mas s f¹ Decreasing Mass

. start - - End Otoserve - ϊ-r( eκpt) l--- ( oa_ o) BeXta Hiss Sequence

► l - - 5 498.36 497.35 496.28 1.08 0 fii?e-?R

1 - - 51 S6S8.18 5697. IB 5697.80 -0.62 4 ---. BPRGIII-S TLϊeiϊSKCBCEROLTCEβBKTWBSITlϊT-WMC-πiaeR

HUI 6 - - 19 1643.17 1642.17 1642.93 -0.77 0 E iιnπ-T X6i∑xκ

20 - - 24 720.61 719.60 720.27 -0.67 0 CBCER

20 - - 51 3594.50 3593.50 3594.61 -1.11 2 C-?CERGLϊC--B»KlrBOSIlHTlϊ-?6ieHDJ-t-R

25 - - 32 873.06 878.06 878.38 -0.32 0 8--TC-.CDK

25 - - 53 3109.00 3108.07 ^■3107.48 0. S9 2 t.LTCBB» Ϊ.WBSIIHTHP(-ICU-)--»-(SK

32 - - 55 51 .18 516.17 517.30 -1.13 1 SKQR con< 32 - - 67 1822.77 1821.76 1822.86 -1.09 2 SKQRSHaPGGHHHHHH

54 - - 55 303.64 302.83 302.17 0.66 0 QR

54 - - 67 1608.79 1607.78 1607.73 0 .05 1 QRSHi-PββMHHHHH sam 54 - - 67 1607.78 1606.77 1S07.73 -0.96 1 βRSH---?GGH-MOfH described in the instant example (Ex. Trypsinization)

First step involved crosslinking a modified human Enterostatin containing a linker (CGGAPGPR) to rabbit serum albumin (Sigma; Saint Louis, MO) using the crosslinking agent bis(sulfosuccinimidyl suberate) (Pierce Biotechnology; Rockport, IL). This was accomplished by slowly adding (dropwise) 1 ml of bis(sulfosuccinimidyl) suberate (5 mg/ml) (BS) in PBS, pH 7.2, to 2 ml of 10 g/L rabbit serum albumin (RSA) in PBS. This solution was stirred for 2 hr at room temperature. Excess bis(sulfosuccinimidyl) suberate was removed by gel filtration in PBS using a 1.6 x 20 cm Sephadex G-50 Column. Protein peaks were identified by monitoring the eluate at 280 nm. Once the protein containing fractions were identified, they were pooled. At this time, 1 mg of Human Enterostatin-ϋnker (CGGAPGPR) was added to the protein peak while gently stirring and incubated overnight at 4^°C. At the end of the incubation period, 100 μl/ml of 1 M Glycine Blocking Solution was added to achieve a final concentration of 0.1 mol/L and gently stirred for a further 2 hr at room temperature. The RSA- BS-APGPR solution was dialyzed (MWCO 10,000 Da) against 0.15 M NaCI for 48 hr at 4^°C with 2 changes of dialysis buffer. At end of dialysis, protein content of solution was determined. The sample was then diluted to a final concentration of 0.5 mg/ml, added sodium azide to a final concentration of 3.1 mM (10.1 πl/ml of a 2% NaN₃ stock solution), aliquoted solution and stored at -20^°C until needed.

Competitive ELISA was performed as follows: 96-well EIA microtitre plate was coated with 100 μl of 0.2 μg/ml RSA-BS-CGG-APGPR or 0.2 μg/ml RSA (blank). The stock solutions was diluted using Coating Buffer (15 mM NaCθ₃/25 mM NaHCOs 3.1 mM NaN₃, pH 9.6) and the plate Incubated overnight at 4^°C. At the end of incubation period, the plates were washed (3X) with Wash Buffer (20 mM Tris HCI/75 mM NaCI/3.1 mM NaNs/0.05% (w/v) Tween 20, pH 7.2-7.4). 100 μl of either unknown or standard APGPR peptide solutions was added followed by 50 μl of 1:2000 rabbit anti-APGPR antisera (Phoenix Pharmaceuticals, Belmont, CA) in ELISA buffer (50 mM Tris HCI/0.05% (w/v) casein/3.1 mM NaNs/10 mM EDTA/0.05% (w/v) Tween 20, pH 7.2-7.4). A dose curve for Enterostatin ranging from 9000 to 0.00073 nM was 5 set up. The plate was further incubated overnight at room temperature. At the end of the incubation period, the plates were washed (3X) with wash buffer (20 mM Tris HCI/75 mM NaGI/3.1 mM aN3/0.05% (w/v) Tween 20, pH 7.2-7.4). 100 μl of goat anti rabbit IgG biotin conjugate (Sigma, St. Louis, MO) (1:1000) in ELISA Buffer was added and incubated for 2 hr at room temperature. The plates were washed (3X) with wash buffer (20 mM Tris HCI/75 mM

10 NaCI/ 3.1 mM NaN3/0.05% (w/v) Tween 20, pH 7.2-7.4), and 100 μl of Extravidin Alkaline Phosphatase solution (Sigma, St. Louis, MO) (1 :500) in wash buffer was added and incubated for 2 hr at room temperature.

The plates were washed (3X) with Wash Buffer (20 mM Tris HCI/75 mM NaCI/3.1 mM NaN₃/0.05% (w/v) Tween 20, pH 7.2-7.4) and 100 μl of 1 mg/ml p-nitrophenyl phosphate

15 substrate buffer (Sigma, St. Louis MO) was added to each well. The plate was incubated until the maximum absorbance for the lowest standard peptide concentration of 1.5 (10 to 25 min at room temperature) was reached. The reaction was terminated by adding 3 mol/L NaOH (50 μl) to each well. The plate was read at 405 nm. Enterostatin concentration of unknowns was calculated based on standard curve generated. Values for the Enterostatin ELISA were as

20 follows: Linear Range: 0.73 to 1500 nM; Intra-Assay CV: 3%; Inter-Assay CV: 4%.

Results of the assay are shown in Figures 8 and 9. This ELISA is highly specific for the APGPR form of enterostatin since the peptides VPDPR and CGGAPRP readings are displaced well to the right of the APGPR standard curve (see Figure 8). In addition, presence 25 of intact CG55698-02 did not interfere with this assay. Serum samples from ten normal human volunteers (males and females) yielded an Enterostatin concentration of 1.57 ± 0.617 m mol/L.

30 Example 4. Confirmation of the Lipase-CG55698-02 Protein Complex Formation and Determination of its Dissociation Constant

The purified mature form of CG55698-02 or its derivative was mixed with pancreatic lipase and the binding was determined using a commercially available Pancreatic Lipase Colorimetric Assay (Pointe Scientific Inc; Lincoln Park, Ml). Competitive binding was also ' 35 confirmed when full-length colipase (CG55698-01) was incubated with pancreatic lipase at the presence of CG55698-02.

Inhibition of Lipase Activity by Binding to CG55698-02 Protein

To demonstrate that splice variant (CG55698-02) and its derivatives can bind to 40 pancreatic lipase and inhibit its activity, varying concentrations of CG55698-02 or its derivatives were mixed with pancreatic lipase. Activity was assessed using a commercially available assay (Pointe Scientific, Inc; Lincoln Park, Ml) developed for the quantitative determination of Pancreatic Lipase in serum. However, in this example this assay is adapted to assess the effects of specific CG55698 proteins (Ex: CG55698-01, CG55698-02) on Pancreatic Lipase activity.

Assay was conducted as described in the manufacturer's instructions (Pointe Scientific, Inc; Lincoln Park, Ml) with the following modifications: A series of microfuge tubes containing equal amount of pancreatic lipase were set up and varying concentrations of CG55698 protein. The reaction was incubated for periods ranging from 5 to 180 min at 37°C. At the end of the incubation period, 5 μl of the reaction mixture was transferred to a 96-well plate. Blanks (distilled water) and standard (pancreatic lipase) were set up as appropriate (depending on the dilution factors). 150 μl of lipase substrate reagent was added to all wells, mixed well and incubated at 37°C for 3 to 5 min. After the pre-incubation, 100 μl of lipase activator was added to each well, mixed and incubated for 3 min at 37°C. The rate of increase in absorbance per minute at 550 nm (540-560 nm) was measured and lipase activity was calculated as follows: ΔAsampie - ΔA_bι_ank / ΔA_std- ΔAbiank) X Standard Concentration (U/L).

Result is shown in Figure 10. Trypsinized CG55698-02, its derivatives and wild type colipase (CG55698-01) were incubated with constant amounts of pancreatic lipase at 37°C. The data show that the trypsinized N-terminal Tagged splice variant did not have any effect on pancreatic lipase activity (Figure 10). On the other hand, trypsinized Wild Type (CG55698- 01) as well as the C-Terminal Tagged splice variant (CG55698-02, both trypsinized and Non- trypsinized) significantly decreased lipase activity. The inhibitory activity of the trypsinized wild type colipase might be related to the generation of inhibitory peptide fragments as this protein is more sensitive to trypsin digestion than the splice variant (CG55698-02; C-Terminal Tagged). These experiments demonstrate the ability of the colipase splice variant to inhibit lipase activity in vitro.

Example 5. CG55698-02 Protein Effects on Weight Control

To demonstrate the efficacy of CG55698-02 polypeptide and its variants in controlling weight gain, animal models such as rat, mouse, or other suitable validated animal model of obesity will be used. The animals will be adapted to either a high fat diet or a two-choice selection of high fat or low fat diets ad libitum. Mature, purified CG55698-02 or any of its variants detailed in the instant specification could be administered orally or parenterally. The time of administration is either before the meal or during the meal. When delivering CG55698-02 protein orally, the protein can be mixed in the food pellet or animals will be gavaged. The protein can be modified or formulated in a variety of pharmaceutical compositions to stabilize the protein, to facilitate intake, or to prolong its action. Acute Studies - CNS Activity

The goal of this study is to demonstrate the central effects of the released Enterostatin peptide, resulting from the action of duodenal trypsin on the splice variant, after oral administration on macronutrient choice.

Male Sprague-Dawley rats (125-150 g) housed individually are randomly allocated to suspended wire mesh cages with water and food provided ad libitum, except as indicated. Rats are adapted to a two-choice high fat (28% protein; 56% energy by fat content; 4.78 kcal/g) and low-fat (22% protein; 10% energy by fat content; 3.66 kcal/g) for one week prior to the start of the experiment. Food is placed in glass cups with special grids or lids to minimize wastage and aid record keeping. The position of the food cups in each cage will be randomly changed each day.

Compounds are administered either by gavage or by intravenous injection. Rats are sham gavaged (0.5 ml) (18 gauge needle; 2.25 mm diameter; 50 mm in length) or sham injected (i.v.; tail vein) daily with saline (0.1 ml) one week prior to the beginning of the study. Body weight, food and water consumption are determined daily during this period.

Rats are adapted to a two-choice high fat (28% protein; 56% energy by fat content; 4.78 kcal/g) and low-fat (22% protein; 10% energy by fat content; 3.66 kcal/g) diets. Both diets are presented concurrently in separate glass cups. A white paper sheet is placed under the cages in order to collect spill food. The position of the food cups in each cage is changed randomly each day. Once body weight and food consumption stabilize, rats are randomly allocated to the various treatment groups (body weight range between 10 to 15 g of the group average). Rats are weighed and fasted for four hours prior to dosing (i.e. tour hours into the dark cycle). Compounds are administered at the beginning of the dark cycle via gavage (0.5ml) (18 gauge needle; 2.25 mm diameter; 50 mm in length) or as an intravenous injection (0.1 ml). A red light is available in the treatment room in order to aid technicians during treatment administration and food consumption measurements. Dosages are corrected for body weight. Following administration of the compounds, food consumption (for each of the diets) and water consumption are determined every 30 min for the first two hours and then hourly for 4 hours. A final measurement is taken 24 hr post-treatment. Once treatment begins, the total duration of the study is 24 hr.

Chronic Studies - CNS/Lipid Absorption - Mouse Diet Induced Obesity (DIO) Model The goal of this study is to demonstrate the central and peripheral effects of

CG55698-02 and its variants, after oral administration on body weight, food and water consumption as well as select metabolic parameters in the mouse model of Diet Induced Obesity (DIO). Male C57BL/6J (~4 weeks, 15-20 g) mice are randomly allocated to polycarbonate cages containing corn chip bedding (2 mice/cage) with food and water provided ad libitum. Starting at 5 weeks of age, male C57BL/6J mice are fed a high fat diet (60% fat; Diet Diet #12492i; Jackson Laboratories) for 5 weeks (total of 74 mice). A group of mice will continue to receive control diet from research Diets (10% fat; D12450Bi) (total of 8 mice) throughout the study. Total number of mice in the study is 82.

Mice will be sham gavaged daily with saline (0.5 ml/50 g) one week prior to the start of treatment (i.e. after four weeks of high fat diet administration). Body weight (individual) and food consumption (cage) is determined every three days throughout the fattening period. Once body weight stabilizes (i.e. no fluctuations), mice with body weights >2 standard deviation of the chow group are randomly allocated to the various treatment groups (Time 0). Body weights will then be determined every three days until the conclusion of the study. Compounds will be freshly mixed at the time of administration.

All compounds are administered daily approximately one hour prior to the onset of the dark cycle (i.e. 9:00 am). Dose administered are corrected to body weight. Once treatment begins, the total duration of the study is 2 weeks with the option to extend for an extra week depending on body weight loss (defined as a statistically significant difference between any of the Dosed Group compared to the High Fat Control Group).

Food consumption and body weight will be monitored throughout the study period.

Metabolic parameters such as glucose, insulin, triglyceride, total cholesterol, LDL cholesterol, HDL cholesterol, will be measured. Circulating level of enterostatin will be monitored using the enterostatin ELISA described before. In addition, fecal fat content will be determined.

Thus we have illustrated and described the preferred embodiment of our invention, it is to be understood that this invention is capable of variation and modification, and we therefore do not wish to be limited to the precis terms set forth, but desire to avail ourselves of such changes and alterations which may be made for adapting the invention to various usages and conditions. Such alterations and changes may include, for different compositions for the administration of the polypeptides according to the present invention to a mammal; different amounts of the polypeptide; different times and means of administration; different materials contained in the administration dose including, for example combinations of different peptides, or combinations of peptides with different biologically active compounds. Such changes and alterations also are intended to include modifications in the amino acid sequence of the specific polypeptides described herein in which such changes alter the sequence in a manner as not to change the functionality of the polypeptide, but as to change solubility of the peptide in the composition to be administered to the mammal, absorption of the peptide by the body, protection of the polypeptide for either shelf life or within the body until such time as the biological action of the peptide is able to bring about the desired effect, and such similar modifications. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims. Having thus described our invention and the manner and process of making and using it in such full, clear, concise and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same.

Claims

We claim

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a. SEQ ID NO: 2, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38; b. mature form of SEQ ID NO: 2, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,

34, 36, 38.

2. The polypeptide of claim 1 wherein the said polypeptide inhibits pancreatic lipase activity in mammals.

3. The polypeptide of claim 2 wherein the said polypeptide reduces side effects associated with lipase inhibition in mammals.

4. The polypeptide of claim 1 wherein the said polypeptide suppresses appetite in mammals.

5. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

6. A kit comprising, in one more containers the composition of claim 5.

7. A method of inhibiting dietary fat absorption in mammals comprising administering the polypeptide of claim 1.

8. A method of reducing side effects associated with lipase inhibition in mammals comprising administering the polypeptide of claim 1.

9. The method of claim 8, wherein the side effects consists of steatorhea, loose faeces, oily spotting, faecal urgency and increased defecation.

10. A method of controlling weight gain in mammals by administering a composition with combined activity of lipase inhibitor and appetite suppressor.

11. The method of claim 10, wherein the composition comprises the polypeptide of claim 1. 12. A method of treating obesity, diabetes, dyslipidemia and cardiovascular disease in mammals comprising administration of polypeptide of claim 1.

13. An isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of: a. a nucleic acid sequence that encodes a polypeptide of amino acid sequence SEQ ID NO: 2, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38; b. a nucleic acid sequence that encodes the mature form of a polypeptide of amino acid sequence SEQ ID NO: 2, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38.

14. A vector selected from a group consisting of yeast, mammalian cell or an insect cell, comprising the polynucleotide of claim 13.

15. The vector of claim 14, wherein a promoter is operably linked to the said polynucleotide.

16. A cell comprising the vector of claim 14.

17. An isolated antibody that immunospecifically binds to the polypeptide of claim 1. 18. The antibody of claim 17 wherein the antibody is a monoclonal antibody.

19. The antibody of claim 17, wherein the antibody is a humanized antibody.

21. A chimeric protein comprising a first polypeptide of claim 1 and a second polypeptide.

22. The chimeric protein of claim 21 , wherein the second polypeptide is fused to the N- terminus or the C-terminus of the first polypeptide of claim 1. 23. The chimeric protein of claim 21 , wherein the second polypeptide is at least a portion of an immunoglobulin molecule. 24. The chimeric protein of claim 21 , wherein the second polypeptide comprises of the Fc region of an immunoglobulin molecule.