WO2002098915A2

WO2002098915A2 - Compositions and methods for detecting or regulating cholesteryl ester transfer protein

Info

Publication number: WO2002098915A2
Application number: PCT/EP2002/006228
Authority: WO
Inventors: Jamila Najib-Fruchart; Zouher Majd
Original assignee: Genfit
Priority date: 2001-06-07
Filing date: 2002-06-06
Publication date: 2002-12-12
Also published as: WO2002098915A3; AU2002325827A1

Abstract

The present invention relates to compositions and methods for detecting or regulating Cholesteryl Ester Transfer Protein ('CETP'). This invention also relates to synthetic CETP peptides, corresponding antibodies, kits comprising the same, and their use to detect, quantify and/or monitor CETP levels in a sample. The above compounds and kits can also be used to modulate CETP levels or activity in vitro or in vivo, and to regulate lipid metabolism in a subject.

Description

Compositions and methods for detecting or regulating Cholesteryl Ester Transfer Protein

The present invention relates to compositions and methods for detecting or regulating Cholesteryl Ester Transfer Protein ("CETP"). This invention also relates to synthetic CETP peptides, corresponding antibodies, kits comprising the same, and their use to detect, quantify and/or monitor CETP levels in a sample. The above compounds and kits can also be used to modulate CETP levels or activity in vitro or in vivo, and to regulate lipid metabolism in a subject.

It is widely accepted that there is a strong inverse relationship between plasma High Density Lipoproteins (HDL) cholesterol concentration and the incidence of Coronary Arterial Disease (CAD) (Gordon 1977 ; Miller 1977). The protective role of HDL against atherosclerosis is mostly related to its function in the so-called reverse cholesterol transport, the process by which excess cholesterol is removed from peripheral tissues, including the arterial wall, and transported to the liver for excretion (Glomset 1968; Fielding and Fielding 1995). In vivo, part of this process involves the esterification of free cholesterol within the HDL fraction by lecithin: cholesterol acyltransferase (LCAT), and its subsequent transfer by cholesteryl ester transfer protein (CETP) to triglyceride-rich lipoproteins.

Human CETP is a 476 amino acids glycoprotein, which belongs to the lipid transfer/lipolysaccharide binding protein family. The complete nucleic acid and amino acid sequence of human CETP is disclosed, for instance, in Day, Albers et al. 1994 (accession number: PI 1597). The apparent molecular weight of CETP is about 74 kDa, with a net molecular weight of 53 kDa. Conflicting data regarding its role in atherosclerosis reflect the intricate balance between the pro- and anti-atherogenic properties of this protein. CETP activity leads to decreased plasma levels of HDL cholesterol (Agellon, Walsh et al. 1991; Marotti, Castle et al. 1993) and increased LDL cholesterol (Marotti, Castle et al. 1993), a lipoprotein profile that may promote atherogenesis. However, CETP can also promote dissociation of apo Al from HDL, generating pre-β-HDL particles (Newnham and Barter 1990; Kunitake, Mendel et al. 1992), which together with the enhanced transport of cholysteryl ester (CE) to triglyceride-rich lipoproteins, may facilitate reverse cholesterol transport.

The absence of atherosclerosis in some CETP-deficient Japanese patients (Inazu, Brown et al. 1990) and the increase in diet-induced aortic atherosclerosis in mice expressing human CETP (Inazu, Brown et al. 1990; Hayek, Masucci-Magoulas et al. 1995) provided support for the initial hypothesis that CETP was a "pro- atherogenic" factor. A recent study demonstrating that infusion of CETP antisense oligonucleotides in cholesterol- fed rabbits inhibited atherosclerosis provided further support for this concept (Sugano, Makino et al. 1998). However, large population studies (Zhong, Sharp et al. 1996; Hirano, Yamashita et al. 1997) have indicated that low CETP plasma levels due to a common genetic polymorphism as well deficiency of CETP are associated with an increased incidence of CAD. In addition, Hayek et al. (Hayek, Masucci-Magoulas et al. 1995) demonstrated that expression of CETP protects against atherosclerosis in hypertriglyceridemic mice. Taken together these data show that plasma levels of CETP can be related to various pathophysiological conditions involved in the atherosclerosis susceptibility and the predisposition to CAD, such as some dyslipoproteinemia. It is thus clearly apparent that the availability of compounds, methods and kits to detect, quantify or modulate CETP levels would be of significant value in the therapeutic, diagnostic, screening and/or experimental areas.

Mainly, two distinct approaches can be applied to the quantification of lipid transfer protein levels, consisting of either evaluation of lipid transfer activities by isotopic or net mass-transfer assays or determination of the mass concentration of lipid transfer proteins by specific immunoassays. Although the determination of lipid transfer rates in plasma has proved helpful and informative, it does not necessarily provide a reliable and specific estimate of the lipid transfer protein mass, due in part to the presence of putative modulators in total plasma, and this technique is not suitable in diagnostic, because it is heavy and time consuming. The result is that only specific immunoassays are suitable for accurate determination of lipid transfer protein mass in plasma samples. In 1990, the first radioimmunoassay of human CETP proposed by Marcel and co-workers (Marcel, McPherson et al. 1990), allowed the determination of mean CETP levels in normolipidemic plasma. However, the latter CETP immunoassay requires antibody labelling and radioisotope handling making it unusable by some laboratories. Other immunoenzymatic assays (ELISA) based on the competitive or non competitive binding of specific mouse anti-CETP were performed, with no need for antibody labelling or radioisotopes, the detection being performed using peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Guyard-Dangremont, Lagrost et al. 1994; Mezdour, Kora et al. 1994). However, these assay systems present the disadvantage to require purified CETP for the production of antibodies and the construction of calibration curves. Additionally the coefficient of variation of all these ELISA is high and the delay for sample measurement is more than one day, because there is a need of several steps of incubations.

The present invention now provides novel compositions and methods for detecting or regulating CETP. The present invention specifically discloses novel methods of producing efficient anti-CETP antibodies using particular synthetic CETP peptides. The invention also discloses such efficient antibodies, kits comprising the same, and their use to detect, quantify, purify and/or monitor CETP levels in a sample. Said antibodies also provide a novel approach to modulate CETP levels or activity in vitro or in vivo, and to regulate lipid metabolism in a subject.

A particular object of this invention relates to polypeptide fragments of CETP and their uses, particularly to produce anti-CETP antibodies. Specific polypeptide fragments of this invention comprise amino acid sequence SEQ ID NO: 1, 2, 3, 4 or 5 or an immunogenic fragment or derivative thereof.

A further object of this invention is a nucleic acid molecule encoding a polypeptide as defined above. A further object of this invention is an antibody that binds a polypeptide as defined above, as well as fragments or derivatives of such antibody.

A further object of this invention is a method of producing an anti-CETP antibody comprising administering (i.e., immunizing) a polypeptide or nucleic acid as defined above to a non-human mammal and collecting the antibody or antibody-producing cells.

A further object of this invention is a method of regulating CETP activity, comprising administering to a subject an antibody (including a fragment or derivative thereof) as defined above.

An other object of this invention is a method of detecting predisposition or individuals at risk of developing lipid-metabolism disorders, comprising detecting in vitro CETP in a sample from a subject with an antibody (including a fragment or derivative thereof) as defined above.

An other object of this invention is a method of monitoring lipid transfer protein mass in a subject, comprising detecting in vitro CETP level in a sample using an antibody (including a fragment or derivative thereof) as defined above.

An other object of this application is a method of regulating the transfer of cholesteryl ester from HDL in a subject, comprising administering to a subject in need thereof, an amount of an antibody (including a fragment or derivative thereof) as defined above effective to regulate cholesteryl ester from HDL. The subject is preferably a mammal, particularly a human, more preferably a subject at risk of developing lipid-disorders such as CAD or a subject having such a disease.

A further object of this invention is a pharmaceutical composition comprising an antibody (including a fragment or derivative thereof) as defined above and a pharmaceutically acceptable excipient or carrier. As indicated above, in a particular aspect, the present invention relates to CETP polypeptide fragments and their use to produce antibodies. Specific polypeptides of this invention have the amino acid sequence of SEQ ID NO: 1 - 5 or an immunogenic fragment or derivative thereof. These polypeptides represent particular synthetic peptides comprising a portion of the amino acid sequence of human CETP or a derivative thereof, selected to improve antibody production. The polypeptides were designed using algorithms to predict flexibility (Westhof, Altschuh et al. 1984; Karplus 1985), hydrophilicity (Kyte and Doolittle 1982; Hoop 1983), antigenicity (Jameson 1988), secondary structure (Chou and Fasman 1978; Gamier, Osguthorpe et al. 1978) and hydrophobic cluster analysis (Lemesle-Varloot, Henrissat et al. 1990).

By using this approach, applicants have identified several domains in human CETP with improved properties. These domains comprise, more specifically, amino acid 368 to amino acid 405 of human CETP (SEQ ID NO:l) and amino acid 346 to amino acid 405 of human CETP (SEQ ID NO: 2).

SEQ ID NO: 1 : Val-Thr-Thr-Val-Gln-Ala-Ser-Tyr-Ser-Lys-Lys-Lys-Leu-Phe- Leu-Ser-Leu-Leu-Asp-Phe-Gln-Ile-Thr-Pro-Lys-Thr-Val-Ser-Asn-Leu-Thr-Glu- Ser-Ser-Ser-Glu-Ser-Ile

SEQ ID NO: 2 : Val-Lys-Phe-Leu-Phe-Pro-Arg-Pro-Asp-Gln-Gln-His-Ser-Val- Ala-Tyr-Thr-Phe-Glu-Glu-Asp-Ile-Val-Thr-Thr-Val-Gln-Ala-Ser-Tyr-Ser-Lys- Lys-Lys-Leu-Phe-Leu-Ser-Leu-Leu-Asp-Phe-Gln-Ile-Thr-Pro-Lys-Thr-Val-Ser- Asn-Leu-Thr-Glu-Ser-Ser-Ser-Glu-Ser-Ile

Other selected polypeptides comprise the following amino acid domains :

28-53 : TAKVIQTAFQRASYPDITGEKAMMLL (SEQ ID NO: 3) 93-122 : VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI (SEQ ID NO: 4)

229-262 : LESHHKGHFIYKNVSEDLPLPTFSPTLLGDSRML (SEQ ID NO: 5)

A particular object of this invention thus relates to a polypeptide comprising amino acid sequence SEQ ID NO: 1, 2, 3, 4 or 5 or an immunogenic fragment or derivative thereof.

The polypeptides of this invention contain preferably less than 100 amino acids, more preferably less than 90 amino acids, even more preferably less than 80 amino acids. The polypeptide more preferably comprises at least an epitope of CETP, even more preferably at least a portion of CETP that includes a hydrophile and flexible domain. Most preferred polypeptides comprise less than 100 amino acids and comprise at least amino acid residues 370-390 of human CETP, which correspond to amino acid residues 3-23 of SEQ ID NO: 1 or 25-45 of SEQ ID NO: 2.

The term "immunogenic fragment" designates any portion of the polypeptide comprising an epitope, preferably a T or B cell epitope. Such fragments contain at least 7 consecutive amino acids of the above polypeptide sequence, even more preferably at least 10 consecutive amino acids of the above polypeptide sequence.

The term "derivative" includes polypeptide comprising one or several mutation, substitution, deletion and/or addition of one or several amino acid residues and retaining substantially the same antigenic specificity. Typical examples of derivatives include sequence variations due to CETP polymoφhism, splicing, etc. Most preferred derivatives contain 5 modified amino acid residues at most, as compared to SEQ ID NO: 1 - 5. Additional residues may correspond to flanking amino acids in the sequence of human CETP, carrier or linker residues, protecting groups, etc. Furthermore, the polypeptides may be modified, for instance by chemical, physical and/or enzymatic modification, to enhance their stability, increase their immunogenicity, incorporate a label or a tracer, facilitate their production or purification, etc. Examples of such modifications include addition of a tag (e.g., myc), a label (e.g., radiolabel, enzymatic label, etc.), a glycosylation, etc.

The polypeptides may be soluble, purified or complexed with a carrier molecule, such as KLH or serum-albumin, for instance or any other inert (e.g., synthetic) molecule, including a bead, etc. They are preferably devoid of natural CETP protein and or of other naturally occurring blood proteins. In a particular embodiment, the polypeptides are coupled to a carrier, especially for use in antibody production. Coupling can be performed according to conventional techniques (Vaitukaitis, Robbins et al. 1971; Bassiri 1979).

The polypeptides may also be conjugated or fused to any heterologous polypeptide molecule, such as a biologically active molecule, for instance. Heterologous designates any polypeptide which does not originate from a human CETP molecule.

Specific examples of a polypeptide of this invention is a polypeptide consisting of SEQ ID NO: 1, 2, 3, 4 or 5, more preferably of SEQ ID NO: 1 or 2.

A further object of this invention is a nucleic acid molecule encoding a polypeptide as defined above. The nucleic acid may be any DNA or RNA molecule, and may further comprise a transcriptional promoter sequence. The nucleic acid may be inserted in a vector (e.g., a plasmid, cosmid, phage, virus, artificial chromosome, etc.). Such nucleic acid constructs can be used to produce the polypeptides in vitro, in any suitable host cell (prokaryotic or eukaryotic) or directly in vivo.

The polypeptides are preferably prepared by synthesis using any conventional method, including solid phase synthesis, as disclosed in Merrifield et al (1963) and in the examples. Artificial synthesis is advantageous since the polypeptides are free of any contaminating compound and can be produced in high quantity and purity.

The polypeptides may be used in screening assays, or in titration assays, as controls, standards or to calibrate the assays. They may also be used to modulate CETP activity. They are also particularly suited to produce anti-CETP antibodies.

In this regards, a further object of this invention resides in an antibody that binds a polypeptide as defined above. Obviously, binding should be specific, meaning that the antibody should not bind specifically to other antigens, and that the binding to other antigens can be discriminated from specific binding to the above CETP peptides. As illustrated in the examples, preferred antibodies of this invention do not bind specifically to albumin or enzymes or distinct lipoproteins. The antibody may be a polyclonal or a monoclonal antibody. Furthermore, the term antibody also includes fragments and derivatives thereof, in particular fragments and derivatives of said monoclonal or polyclonal antibodies having substantially the same antigenic specificity. These include antibody fragments (e.g., Fab, Fab'2, CDRs, etc), humanized antibodies, poly-functional antibodies, Single Chain antibodies (ScFv), etc. These may be produced according to conventional methods, including immunization of an animal and collection of serum (polyclonal) or spleen cells (to produce hybridomas by fusion with appropriate cell lines).

Methods of producing polyclonal antibodies from various species, including mice, rodents, primates, horses, pigs, rabbits, poultry, etc. may be found, for instance, in Vaitukaitis et al., 1971. Briefly, the antigen is combined with an adjuvant (e.g., Freud's adjuvant) and administered to an animal, typically by sub-cutaneous injection. Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separared.

Methods of producing monoclonal antibodies from various species as listed above may be found, for instance, in Harlow et al (Antibodies: A laboratory Manual, CSH Press, 1988) or in Kohler et al (Nature 256 (1975) 495), incoφorated therein by reference. Briefly, these methods comprise immunizing an animal with the antigen, followed by a recovery of spleen cells which are then fused with immortalized cells, such as myeloma cells. The resulting hybridomas produce the monoclonal antibodies and can be selected by limit dilutions to isolate individual clones. Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al (Nature 341 (1989) 544).

Preferred antibodies of this invention are prepared by immunization with a fragment of a CETP polypeptide as described above, preferably with a peptide of SEQ ID NO: 1, 2, 3, 4 or 5 or an immunogenic sub-fragment thereof, e.g., a subfragment comprising at least an epitope, preferably of at least 5 amino acids.

Further preferred antibodies of this invention are monoclonal antibodies that specifically bind an epitope comprised in SEQ ID NO: 1 or 2 or 3 or 4 or 5. Fab or F(ab')2 fragments may be produced by protease digestion, according to conventional techniques. Humanized antibodies can be prepared as previously described (Jones 1986; Riechmann 1988).

This invention also relates to a method of producing an anti-CETP antibody, comprising injecting a polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5, or an immunogenic fragment or derivative thereof, to an non-human animal and collecting the antibodies or antibody-producing cells. The method is simpler than previously disclosed methods using full length natural human CETP and allows the production of specific and immunoprecipitating antibodies. The specificity can be verified by showing the absence of cross-reactivity with other blood proteins such as apolipoproteins, LCAT or lipoprotein lipase (LPL). More generally, the specificity indicates that the antibodies bind CETP with a higher affinity than other antigens. As illustrated in the examples, polyclonals of this invention are immunoprecipitating and can thus be used to detect or dose CETP with high efficacy. The antibodies may be coupled to heterologous moieties, such as toxins, labels, drugs or other therapeutic agents, covalently or not, either directly or through the use of coupling agents or linkers. Labels include radiolabels, enzymes, fluorescent labels, magnetic particles and the like. Toxins include diphteria toxins, botulinum toxin, etc. Drugs or therapeutic agents include lymphokines, antibiotics, antisense, growth factors, etc. Methods of using such heterologous moieties are illustrated, for instance, in US4,277,149 and US3,996,345.

The antibodies of this invention have various applications, including therapeutic, diagnostic, purification, detection, prophylactic, etc.

In vitro, they can be used as screening agents or to purify the antigen from biological samples (e.g., plasma or serum samples). They can also be used to detect or quantify the presence (or amounts) of CETP in a sample collected from a subject, typically a blood sample from a mammalian, specifically a human subject.

In this regard, an other object of this invention is a method of detecting CETP in a biological sample, comprising contacting the sample with an antibody as defined above (including fragments or derivatives thereof) and detecting the presence of antibody-antigen immune complexes. Typically the method allows the determination of the levels of CETP is a sample, by assessing the (relative) amounts of immune complexes in the sample and comparing the same to a standard condition or a calibration curve, for instance. The method may be performed using any conventional technique, such as ELISA, RIA, EIA, any sandwich immuno-assay or direct immunoassay, etc. More preferably, the method is a nephelometric assay. Indeed, as indicated above, the antibodies are specific and can immunoprecipitate CETP in a sample. In the nephelometric assay, the intensity of light scattered by particles in suspension is measured using an analyser. The particles are formed by the immunoprecipitation reaction that occurs in a polymer-enhancing buffer when a specific antibody is brought into contact with the specific antigen. The complexing of an antigen with an antibody specific for the antigen occurs at a rate which increases gradually at first, then rapidly, and finally proceeds through a peak value that is proportional to the antigen concentration. The assay is based on a measure of the maximum rate of change from the scattered light signal, which is correlated (and can be converted) to the antigen concentration. Typically, the nephelometric assay is perfomed using using Beckman immunochemistry systems (IMMAGE), which presents the results on the alphametric display. The nephelometric assay of this invention is advantageous since it is rapid and reproducible and can be implemented on a high throuput basis. Indeed, this assay is performed in a few seconds only for each sample, versus one day in most prior art techniques, and the coefficient of variation is 4 % only versus more than 10 % for CETP detection assays described in the prior art.

A particular object of this invention thus lies in a method of detecting CETP in a biological sample, comprising contacting the sample (or a dilution thereof) with an antibody as described above (including fragments or derivatives thereof), typically at various dilutions thereof, and assessing the formation of CETP-antibody immune complexes by nephelometric assay. More preferably, the antibody is subjected to a treatment prior to being contacted with the sample, in order to remove non-immunoglobulin proteins and/or to concentrate the antibody. The treatment typically comprises contacting the antibodies with polyethylene glycol (PEG), as described for instance in Ritchie et al., 1972. Typically, from 0.5 to 1 μg of specific antibodies are used in the assay, although the skilled person may use different quantities without departing from the instant invention.

In a nephelometric assay, polyclonal antibodies are generally used. In other assay formats (ELISA, etc.), monoclonals may be used as well. The method can be carried out on various biological samples, including plasma, serum, interstitial fluid, urine samples, etc. The sample may be collected from a subject (e.g. a human subject) and used directly for the assay.

Alternatively, the sample may be diluted and/or stocked (for instance in frozen state) for later testing.

The detection assay can be used in various experimental, clinical and/or diagnostic conditions.

In particular, the method can be used to detect predisposition or individuals at risk of developing lipid-metabolism disorders, particularly CAD. A particular object of this invention is a method of detecting predisposition or individuals at risk of developing lipid-metabolism disorders, comprising detecting in vitro CETP in a sample from a subject with an antibody as defined above (including fragments or derivatives thereof), wherein deregulated CETP levels (as compared to as compared to a mean value of a normal subjects) are indicative of individuals at risk of developing lipid metabolism disorders. Typically, a significant deregulated CETP level is an increase or a decrease of at least 20%, preferably at least 50% over the standard situation or mean value, or the absence of the CETP protein in patients. An other object of this invention is a method of monitoring lipid transfer protein mass in a subject, comprising detecting in vitro CETP level in a sample using an antibody as defined above(including fragments or derivatives thereof).

An other object of this invention is a method of monitoring the efficacy of a lipid-metabolism-related disorder treatment in a subject comprising detecting CETP levels in vitro in a sample from said subject using an antibody as defined above (including fragments or derivatives thereof), after administration of said treatment to the subject. Typically, the efficacy of the treatment is correlated to the CETP activity or level in the subject. The efficacy can be correlated to the ability of the treatment to regulate CETP level or activity or to restore normal CETP activity or level in a subject. A further object of this invention is a method of evaluating the physiological state of a subject, e.g., the lipid metabolism activity or level in a subject, comprising detecting CETP levels in vitro in a sample from said subject using an antibody as defined above (including fragments or derivatives thereof).

The antibodies can also be used to screen compounds or diets that might modulate CETP concentration in serum. Typically, the method comprises admimstering a compound or subject an animal or patient to a diet, collecting a biological sample from the animal or patient and detecting or dosing CETP level in said sample using an antibody as defined above (including fragments or derivatives thereof).

As indicated above, these methods can be carried out on various samples (typically plasma or serum) and can be performed by ELISA, RIA, EIA, etc., most preferably by nephelometric assay.

The antibodies of this invention may also be used for the preventive or curative treatment of various pathological conditions, particularly lipid disorder- related diseases. Indeed, as indicated above, CETP levels and activity can be related to various pathophysiological alterations involved in atherosclerosis susceptibility and predisposition to CAD. The antibodies of this invention can be used to regulate CETP levels and/or activity in vivo, and thus interfere with the above pathological conditions.

A further object of this invention relates to the use of an antibody as defined above (including fragments or derivatives thereof) in the manufacture of a composition for regulating CETP activity in vivo in a subject. Another object of this invention is a method of regulating CETP activity in a subject in need thereof, comprising administering to a subject an antibody as defined above (including fragments or derivatives thereof). An other object of this application is a method of regulating the transfer of cholesteryl ester from HDL in a subject, comprising administering to a subject in need thereof, an amount of an antibody as defined above (including fragments or derivatives thereof) effective to regulate cholesteryl ester from HDL. The invention also relates to the use of an antibody as defined above (including fragments or derivatives thereof) in the manufacture of a composition for regulating the transfer of cholesteryl ester from HDL in a subject.

The subject is preferably a mammal, particularly a human, more preferably a subject at risk of developing lipid-disorders such as CAD or a subject having such a disease.

A further object of this invention is a pharmaceutical composition comprising an antibody as defined above (including fragments or derivatives thereof) and a pharmaceutically acceptable excipient or carrier. The composition typically comprises between about 0,01 μg and 1000 μg of polyclonal or monoclonal antibody. More preferably, the antibody is a monoclonal antibody or a derivative thereof, such as a humanized or ScFv antibody.

The pharmaceutical composition may be used for the curative or preventive treatment of various lipid metabolism disorders .

The composition may be administered by injection in various sites, such as sub-cutaneous, intra-veinous, intra-arterial, infra-muscular, intra-dermic, etc. The carrier includes any saline solution, isotonic buffer, including any conventional stabilizer, etc. The composition may include any additional active agent, for use in combination with the antibodies, either simultaneously, or separated or sequentially.

This invention also relates to a kit comprising a polypeptide or an antibody as described above. The kit can be used to detect or quantify CETP in any sample. Further aspects and advantages of this invention will be disclosed in the following examples, which should be regarded as illustrative and not limiting the scope of this application.

Legend to the Figures

Figure 1: Specificity of the Antibodies Figure 2 : Nephelometric CETP assay

Examples

1. Selection of the appropriate polypeptide sequences.

The polypeptide fragments of CETP were selected using algorithms to predict flexibility, hydrophilicity, antigenicity, secondary structure and hydrophobic cluster analysis.

Preferred selected domains comprise amino acid 346 to amino acid 405 or amino acid 368 to amino acid 405 of human CETP (SEQ ID NO: 2 and 1, respectively). Additional selected domains consist of SEQ ID NO: 3, 4 and 5.

2. Polypeptide synthesis

The polypeptides were synthesized by the solid phase method (Merrifield 1963) on an automated synthesizer Model ABI 431 A (Applied Biosystems Inc.) using Boc/Bzl strategy on 0.5 mmol of MBHA resine. Each amino acid was coupled twice by dicyclohexylcarbodiimide/hydroxybenzotriazole without capping. The crude products were purified and analysed by reversed-phase HPLC on a Vydac C18 column using linear gradient from 0 to 100% Buffer B (Buffer A: 0.05% TFA in H₂0 and Buffer B: 0.05% TFA, 60% CH₃CN in H₂0). The molecular masses were determined using an API (Perkin-Elmer) of a simple quadrupole ion electrospray mass spectrometer equipped with an ion -spray (nebulizer-assisted electrospray) source (SCiex, Toronto, Canada). Amino acid analyses were performed using Beckman 6300 amino acid analyser (Beckman instruments, Fullerton, CA), after hydrolysis with 6N HC1 containing 0.25 % phenol at 110°C for 24 h.

The peptides were coupled to a protein carrier (KLH) for the immunization step.

3. Immunization

Anti-sera to CETP polypeptides were prepared in rabbits essentially as described earlier (Vaitukaitis, Robbins et al. 1971). Each polypeptide was emulsified in complete Freud's adjuvant and injected sub-cutaneously to rabbits using 0.5 mg peptide per injection for the first two injections, followed at 15 day intervals with boosters in the same adjuvant but using only 0.25 mg of peptide. Anti-sera were collected as described. Immunization may be carried out using each polypeptide separately or various combinations thereof.

4. Isolation of immunoglobin (Ig G) anti- peptide CETP

Ig G were prepared by modified protocol of Ritchie et al (Ritchie 1972). In particular, non immunoglobulin proteins were removed from immune-serum and the total IgG are dialysed and concentrated.

5. Specificity of the antibody.

Analytical immunoblot of HDL from 4 normolipemic subjects was performed to assess the specificity of anti CETP seq 1 and anti CETP seq 2. As shown in Figure 1, no cross-reaction was observed with the other proteins (enzymes, albumin, apolipoproteins) when the two anti-CETP polypeptides were used as detecting antibodies.

6. Immuno-nephlemetric assay

Reagents and materials

The reagents and materials are listed in the following Table 1.

Table 1

Anti-CETP IgG:

Anti CETP IgG as produced in examples 3 and 4 above are ready to use. They can be refrigerated at 2 to 8°C for use in the week, or frozen at -20°C for use up to months. The immunoglobulins contain sodium azoture.

Standard: CETP standard is a human serum pool, tested for HIV and hepatitis viruses, handled according to the usual precautions in order to prevent contamination. Standard CETP level is 0.213 mg/dl. For constructing the calibration curve, the standard is diluted as indicated in Table 2 below:

Table 2

Sample preparation:

Fresh or frozen (-80°C) samples are recommended for analysis. Sera are collected according to established procedures in clinical laboratory testing. If needed, samples can be kept frozen for longer storage periods; frozen samples are stable for up to one year. Prior to use, the samples are diluted 3 fold in the diluent 1.

Protocol:

. Program a user-defined reagent with the parameters listed above, according to the IMMAGE Immunochemistry System Operations Manual. . Transfer antibody reagent to compartment A of a new User Defined Cartridge. . Put the buffer 1 in compartment buffer.

. Enter the calibrator value from the standard (actual standard CETP value is 0.213 mg/dl) in parameter table according to the dilution scheme shown in table 2. . Use Diluent 1 as the sample diluent. Summary

Chem Name CETP Units mg/dl

Lot Number See Cartridge Protocol Non-Competitive Nephelometric

Reagent Serial See Cartridge Reagent Expiration To be defined by the user

Sample or 21 μl Gain 3

Dilution Volume

Reagent Buffer 195 μl Cal Dilution 1/1

Volume

Compartment 10 μl Sample Dilution 1/1*

Volume

Compartment O μl Reaction time xxxx

Volume

* Manual samples dilutions.

Results

The results are presented on Figure 2. The coefficient of the variation of the method is 4%. The method allows efficient and rapid determination of CETP levels in any biological sample.

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Claims

REVENDICATIONS

1. A polypeptide of less than 100 amino acids, comprising amino acid sequence SEQ ID NO: 1, 2, 3, 4 or 5 or an immunogenic fragment or derivative thereof.

2. The polypeptide of claim 1, comprising at least 7 consecutive amino acids of SEQ ID NO: 1, 2, 3, 4 or 5, more preferably at least 10 consecutive amino acids thereof.

3. The polypeptide of claim 1 or 2, wherein the polypeptide is soluble or complexed with a carrier molecule, such as KLH, serum-albumin or a bead.

4. A polypeptide consisting of SEQ ID NO: 1, 2, 3, 4 or 5.

5. A nucleic acid molecule encoding a polypeptide of any one of claims 1 to 4.

6. An antibody that binds a polypeptide of any one of claims 1 to 4 or a fragment or derivative of said antibody having substantially the same antigenic specificity.

7. The antibody of claim 6, wherein the antibody is produced by immunizing an animal with a polypeptide of any one of claims 1 to 4 and collecting the antibody or antibody-producing cells.

8. The antibody of claim 6 or 7, which is a polyclonal antibody.

9. The antibody of claim 6 or 7, which is a monoclonal antibody.

10. A method of producing an anti-CETP antibody, comprising injecting a polypeptide of any one of claims 1 to 4 or a nucleic acid of claim 5 to an non- human animal and collecting the antibodies or antibody-producing cells.

11. A method of detecting CETP in a biological sample, comprising contacting the sample with an antibody of any one of claims 6 to 9 and detecting the presence of antibody-antigen immune complexes.

12. The method of claim 11, wherein the presence of antibody- antigen immune complexes is determined by ELISA, RIA, sandwich immuno-assay or direct immunoassay.

13. The method of claim 11, wherein the presence of antibody-antigen immune complexes is determined by nephelometric assay.

14. A method of detecting CETP in a biological sample, comprising contacting the sample with an antibody of any one of claims 6 to 9 and assessing the formation of CETP-antibody immune complexes by nephelometric assay.

15. The method of any one of claims 11 to 14, wherein the biological sample is a blood sample or a serum sample.

16. A method of detecting predisposition or individuals at risk of developing lipid-metabolism disorders, comprising detecting in vitro CETP in a sample from a subject with an antibody of any one of claims 6 to 9.

17. A method of monitoring lipid transfer protein mass in a subject, comprising detecting in vitro CETP level in a sample using an antibody of any one of claims 6 to 9.

18. A method of monitoring the efficacy of a lipid-metabolism-related disorder treatment in a subject comprising detecting CETP levels in vitro in a sample from said subject using an antibody of any one of claims 6 to 9.

19. Use of an antibody of any one of claims 6 to 9 to screen in vitro compounds or diets that modulate CETP concentration in serum.

20. An antibody of claim 8 or 9, wherein said antibody is coupled to a heterologous moiety, such as a toxin, label, drug or other therapeutic agent.

21. Use of an antibody of any one of claims 6 to 9 or 20 in the manufacture of a composition for regulating CETP activity in vivo in a subject.

22. Use of an antibody of any one of claims 6 to 9 or 20 in the manufacture of a composition for regulating the transfer of cholesteryl ester from HDL in a subject.

23. A pharmaceutical composition comprising an antibody of any one of claims 6 to 9 or 20 and a pharmaceutically acceptable excipient or carrier.

24. A kit comprising a polypeptide of any one of claims 1 to 4 or an antibody of any one of claims 6 to 9 or 20.